An Investigation Into the Heterogeneity of Insect Arylalkylamine N-Acyltransferases

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11-4-2019

An Investigation into the Heterogeneity of Insect Arylalkylamine N-Acyltransferases

Brian G. O'Flynn University of South Florida

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An Investigation into the Heterogeneity of Insect Arylalkylamine N-Acyltransferases

Brian G. O’Flynn

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida

Major Professor: David J. Merkler, Ph.D. Bill J. Baker, Ph.D. Ioannis Gelis, Ph.D. Yu Chen, Ph.D.

Date of Approval: October 16, 2019

Keywords: AANAT, Insecticide, Acetylation, Fatty-acid Amide, Tribolium castaneum

“In order to be irreplaceable, one must always be different”

Coco Chanel

ACKNOWLEDGEMENTS

I would first thank Dr. David Merkler, for his tireless mentorship and guidance throughout my time at USF. The wisdom and knowledge he has shared with me have been invaluable in every aspect of my life. I can only hope I have returned his support in part through my work here and can look forward to the next stages of our affiliation as colleagues and friends. Further, I would like to thank my committee members, Dr. Ioannis

Gelis, Dr. Yu Chen, and Dr. Bill Baker, for all the collaborations and beneficial discussions shared over the years. I am especially grateful to Dr. Baker, for offering me the opportunity to join USF Chemistry, back when I was a directionless undergraduate.

I would also like to thank all the members of the Merkler lab, past and present.

Without your advice, inspiration, and friendship, I would not have made it this far. This is especially true for Dr. Ryan Anderson and Gabby Suarez. The times we spent together, in lab and out, were unforgettable, and made even the toughest days better. I also had the great fortune to mentor several incredible undergraduates. The countless hours they devoted to their research became the backbone of this dissertation. I thank you all for your tireless contribution, especially Karin Claire Prins and Britney Shepherd, for who I am grateful not just for your amazing work ethic, but also the positivity you brought to the lab. Thank you also to Dr. Richard Pollenz and the STEM Academy. Being a graduate mentor was one of the most amazing experiences of my life, and I look forward to the exciting future of this program.

To all my friends in Ireland, thank you for always being there for me. I will never take for granted the feeling of going home and feeling like I never left. To those in Florida who welcomed me so warmly, I am eternally grateful. A special mention must be given to

Periodically Right, and all those trivia nights and tailgates that kept me sane and social.

To Briana, words cannot describe the impact you have had in my life. You have been my best friend, and I can only hope I make you half as proud of me as I am of you.

Finally, I would like to thank my family for their constant support. To Mam and Dad, you have always been there for me, and I will never be able to repay all you have given.

But I will try. To Lisa and Keith, thank you for being my benchmark for how to get the most out of life. I will never stop looking up to you. To Nanny and aunt Geraldine, may you rest in peace. I carry your memory with me always.

TABLE OF CONTENTS

LIST OF TABLES ...... iv

LIST OF FIGURES ...... vi

ABSTRACT ...... ix

CHAPTER ONE: INTRODUCTION AND MOTIVATION ...... 1 1.1 Enzyme heterogeneity ...... 1 1.2 A brief history of pesticides ...... 3 1.3 Insecticide resistance ...... 5 1.4 AANATs as a potential insecticide target ...... 7 1.5 References ...... 15

CHAPTER TWO: INSECT AANATS: MECHANISMS AND ROLE IN FATTY ACID AMIDE BIOSYNTHESIS ...... 26 2.1 Introduction ...... 26 2.2 N-Acetylation of the aromatic acids and aromatic amino acid – derived metabolites ...... 29 2.3 Insect AANAT – Kinetic mechanism ...... 34 2.4 Insect AANAT – Catalytic mechanism ...... 36 2.5 Insect AANAT – a “timezyme”? ...... 47 2.6 Conclusion ...... 49 2.7 References ...... 50

CHAPTER THREE: IDENTIFICATION OF CATALYTICALLY DISTINCT AANAT SPLICOFORMS FROM TRIBOLIUM CASTANEUM ...... 68 3.1 Introduction ...... 68 3.2 Results and discussion ...... 70 3.2.1 Amine screening ...... 70 3.2.2 Amine steady-state kinetic constants ...... 71 3.2.3 Acyl-CoA steady-state kinetic constants ...... 73 3.2.4 Importance of the AANAT N-terminus ...... 74 3.3 Conclusion ...... 78 3.4 Materials and methods ...... 78 3.4.1 Cloning of TcAANAT1b ...... 78 3.4.2 Screening assay ...... 79 3.5 References ...... 82 i

CHAPTER FOUR: CHARACTERIZATION OF AANAT FROM TRIBOLIUM CASTANEUM: A POTENTIAL NEXT-GENERATION INSECTICIDE TARGET ...... 89 4.1 Introduction ...... 89 4.2 Results and discussion ...... 92 4.2.1 Acyl-CoA steady-state kinetic constants ...... 92 4.2.2 Amino steady-state kinetic constants ...... 93 4.2.3 Product characterization ...... 94 4.2.4 TcAANAT0 kinetic mechanism ...... 95 4.2.5 Crystal structure and mutagenesis of TcAANAT0 ...... 103 4.2.6 TcAANAT0 chemical mechanism ...... 107 4.3 Conclusion ...... 112 4.4 Experimental procedures ...... 112 4.4.1 Materials ...... 112 4.4.2 Cloning of T. castaneum AANAT0 ...... 113 4.4.3 Determination of enzyme activity ...... 114 4.4.4 Determination of kinetic mechanism ...... 114 4.4.5 Determination of acetyl-CoA binding constants ...... 117 4.4.6 Determination of pH-dependence ...... 118 4.4.7 Product characterization ...... 119 4.4.8 TcAANAT0 mutagenesis...... 119 4.4.9 TcAANAT0 crystallization ...... 120 4.4.10 X-ray data collection and refinement ...... 120 4.5 References ...... 120

CHAPTER FIVE: Bm-iAANAT3: EXPRESSION AND CHARACTERIZATION OF A NOVEL ARYLALKYLAMINE N-ACYLTRANSFERASE FROM BOMBYX MORI ..... 130 5.1 Introduction ...... 130 5.2 Results and Discussion ...... 131 5.2.1 Cloning, expression, and purification of Bm-iAANAT3 ...... 131 5.2.2 Identification of amine and acyl-CoA substrates for Bm-iAANAT3 ...... 132 5.2.3 Kinetic mechanism ...... 137 5.2.4 pH dependence and identification of a catalytic base ...... 142 5.2.5 Identification of a catalytic base and a potential chemical mechanism for catalysis ...... 144 5.3 Conclusion ...... 147 5.4 Experimental procedures ...... 148 5.4.1 Materials ...... 148 5.4.2 Cloning of Bm-iAANAT3 ...... 148 5.4.3 Substrate identification for Bm-iAANAT3 ...... 148 5.4.4 Determination of the steady-state kinetic constants ...... 149 5.4.5 Kinetic mechanism for Bm-iAANAT3 ...... 150 5.4.6 Cloning, expression, and purification of site-directed mutants ...... 153 5.5 References ...... 153

CHAPTER SIX: WHAT REMAINS TO BE UNEARTHED? ACTIVE-SITE DISORDER HOLDS KEY TO LONG-CHAIN ACYLATION BY AANAT ...... 164 6.1 Introduction ...... 164 6.2 AANATs to fill the gap? ...... 165 6.3 Long-chain acyltransferase activity in AANATs ...... 167 6.4 Phylogenetic and Structural insight into long-chain acylation ...... 170 6.5 Conclusion ...... 174 6.6 References ...... 175

APPENDIX A: REPRODUCTION PERMISSIONS ...... 186

APPENDIX B: SUPPELEMENTARY INFORMATION ...... 193

iii

LIST OF TABLES

Table 1.1: KM values for the iAANATs found in D. melanogaster and B. mori with respect to different acyl chain lengths ...... 10

Table 1.2: Binding constants for iAANATs found in D. melanogaster and B. mori with respect to different amine acceptors ...... 11

Table 3.1: Screening results for three distinct pools of amines ...... 70

Table 3.2: Steady-state kinetic constants of most active amine substrates for TcAANAT1b ...... 71

Table 3.3: Kinetic constants recorded for different variants of TcAANAT1...... 72

Table 3.4: Steady-state kinetic constants of acyl-CoA substrates for TcAANAT1b ...... 73

Table 4.1: Steady-state kinetic constants of active acyl-CoA substrates for TcAANAT0 ...... 93

Table 4.2: Steady-state kinetic constants of most active amine substrates for TcAANAT0 ...... 94

Table 4.3: TcAANAT0 product characterization ...... 95

Table 4.4: Inhibition patterns observed for inhibitors of the amine binding pocket (tryptophol) and acyl-CoA binding pocket (desulfo-CoA) ...... 99

Table 4.5: Summary of kinetic rate and inhibition constants attributed to time-dependent inhibition ...... 102

Table 4.6: Steady-state kinetic constants for TcAANAT0 site-directed mutants .. 107

Table 4.7: Summary of pKa values measured from pH rate profiles...... 109

Table 5.1: Pooled amine substrates for the initial screening assay ...... 133

Table 5.2: Kinetic constants for the amine substrates for Bm-iAANAT3 ...... 134 iv

Table 5.3: Kinetic constants for the CoA thioester substrates (R-CO-S-CoA) for Bm-iAANAT3 ...... 135

Table 5.4: Ratio of (kcat,wildtype)/(kcat,mutant) for general base mutant enzymes ...... 145

Table 6.1: Summary of kinetic data obtained for Bm-iAANAT and DmAANATL2 utilizing acyl-CoAs of varying chain-length ...... 167

Table 6.2: Summary of kinetic data obtained for TcAANAT0 and TcAANAT1b utilizing acyl-CoAs of varying chain-length ...... 169

Table 6.3: Summary of kinetic data obtained for DmAANATA, Bm-iAANATL3 and DmAgmNAT utilizing acyl-CoAs of varying chain-length ...... 169

LIST OF FIGURES

Figure 1.1: The hypothesized process of enzyme evolution...... 2

Figure 1.2: Proposed mechanism of pesticide resistance ...... 6

Figure 1.3: Mechanism for the AANAT-catalyzed N-acetylation of amines and arylalkylamines ...... 7

Figure 1.4: Primary sequence alignment of various insect AANATs, as well as human serotonin N-acetyltransferase (SNAT)...... 13

Figure 2.1: Neighbor-joining tree on characterized insect arylalkylamine N- acetyltransferases (iAANATs) ...... 29

Figure 2.2: The reaction catalyzed by arylalkylamine N-acyltransferase...... 31

Figure 2.3: The reaction catalyzed by NBAD synthase ...... 33

Figure 2.4: Sequence alignment of iAANATs ...... 39

Figure 2.5: Crystal structure of DmAANATA active site with bound acetyl-CoA ...... 40

Figure 2.6: Crystal structure of DmAANATA active site with bound acetyl-CoA aligned with apo structure of DmAgmNAT ...... 41

Figure 2.7: Crystal structure of DmAANATA active site with bound acetyl- CoA aligned with homology models of DmAANAT2 and DmAANATL7 ...... 42

Figure 2.8: The importance of the active site histidine of iAANATs in the structural maintenance of the active site ...... 44

Figure 2.9: Proposed catalytic mechanisms for iAANAT...... 46

Figure 3.1: Sequence alignment of AANAT1, AANAT1b and AANAT0 from Tribolium castaneum, as well as AANATA from Drosophila melanogaster ...... 75

Figure 3.2: Structure of SNAT with and without bisubstrate analog bound ...... 77

Figure 3.3: Reaction of CoA-SH with 4,4’-dithiodipyridine producing a conjugated thioketone detectable at 324 nm (Ɛ = 19,800 M-1cm-1 at pH 8.0) ...... 80

Figure 4.1: Acetyl-CoA and tryptamine initial velocity double reciprocal plots ...... 96

Figure 4.2: ITC measurements of acetyl-CoA binding to TcAANAT0 ...... 97

Figure 4.3: Inhibition patterns observed for desulfo-CoA for the amine binding pocket and acyl-CoA binding pocket...... 99

Figure 4.4: Inhibition patterns observed for tryptophol of the amine binding pocket and acyl-CoA binding pocket...... 99

Figure 4.5: Inhibition patterns observed for CoA-S-acetyltryptamine ...... 100

Figure 4.6: Time-dependent inhibition of TcAANAT0 by CoA-S-acetyltryptamine ...... 101

Figure 4.7: Kinetic model for slow-binding inhibition ...... 102

Figure 4.8: Crystal Structure of TcAANAT0 in complex with acetyl-CoA ...... 104

Figure 4.9: Structure of acetyl-CoA binding interactions within the TcAANAT0 active site ...... 105

Figure 4.10: Primary sequence alignment showing conserved Glu residue ...... 106

Figure 4.11: pH rate profiles for DFPE illustrating the effect of pH on kcat,and kcat/KM ...... 108

Figure 4.12: pH rate profiles for tryptamine illustrating the effect of pH on kcat, and kcat/KM...... 108

Figure 4.13: pH rate profiles for acetyl-CoA illustrating the effect of pH on kcat, and kcat/KM...... 108

Figure 4.14: Proposed chemical mechanism for T. castaneum AANAT0 with an unidentified general acid responsible for the identified pKa of 9.8 ...... 110

Figure 4.15: Proposed chemical mechanism for T. castaneum AANAT0 with the departing CoA-SH responsible for the identified pKa of 9.8...... 111

vii

Figure 5.1 Homology overlays of iAANAT enzymes from Bombyx mori and Drosophila melanogaster ...... 132

Figure 5.2: Bm-iAANAT3 double-reciprocal analysis of initial velocities for acetyl-CoA and tryptamine ...... 138

Figure 5.3: Dead-end inhibition plots for Bm-iAANAT3 ...... 140

Figure 5.4 Product inhibition of Bm-iAANAT3 by N-acetyltryptamine ...... 141

Figure 5.5: pH rate profiles for Bm-iAANAT3...... 143

Figure 5.6: Proposed mechanism for the Bm-iAANAT3-catalyzed N-acetylation of tryptamine...... 146

Figure 6.1: Schematic demonstrating the inherent spectrum of acylation utilized by various insect AANATs based off their obtained kinetic data ...... 170

Figure 6.3: Overlay of crystal structure obtained for DmAANATA with homology model of DmAANATL2 ...... 173

viii

ABSTRACT

Arylalkylamine N-acyltransferases (AANATs) have, in recent years, been suggested as potential new insecticide targets. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. Mammalian AANAT is predominantly associated with circadian rhythm regulation, as it catalyzes the formation of N-acetylserotonin, the precursor of melatonin, from serotonin. In insects, this process is a key step in melanism, as well as hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each insect AANAT

(iAANAT) isoform characterized indicates that while catalyzing similar reactions, each insect genome contains distinct iAANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology, and would make iAANATs a valuable target to combat insecticide resistance. As highlighted by the World Health Organization, however, to develop an effective resistance management plan (RMP), intrinsic biological knowledge of existing and potential targets is vital to understand the development of resistance.

This dissertation is dedicated to the consolidation and expansion of our knowledge of iAANATs both mechanistically and structurally. Significant efforts have thus far been put in to characterizing this group of enzymes and their products in several different insects. These efforts are highlighted and are built upon with the identification and characterization of three novel iAANATs: TcAANAT1b and TcAANAT0 from Tribolium ix castaneum, and Bm-iAANAT3 from Bombyx mori. TcAANAT1b is a truncated form of the putative dopamine N-acetyltransferase of T. castaneum: TcAANAT1. This shortened

‘splicoform’ was shown to be a significantly more active form of the enzyme, indicating a catalytic importance of the N-terminus of iAANATs. TcAANAT0 was shown to be a much more promiscuous enzyme, catalyzing the formation of short chain N-acylarylalkylamines via ordered sequential mechanism, with short-chain acyl-CoAs (C2-C10) functioning in the role of acyl-donor. The first crystal structure was also obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. Bm-iAANAT3 was found to be a “versatile generalist”, functioning via a random kinetic mechanism. pH-rate profiles generated for both enzymes indicated key catalytic residues which were used to solve the chemical mechanism of both TcAANAT0 and Bm-iAANAT3. Both these enzymes were also found to catalyze the formation of succinylated and malonated amides respectively, indicating a potential panel of biologically occurring N-dicarboxylated amines awaiting discovery. However, none of the above enzymes were found to catalyze the formation of long-chain fatty acid amides, however. The previous characterization of two enzymes, Bm-iAANAT and DmAANATL2, which do perform this role in vitro indicates that some evolutionary divergence took place to separate short and long-chain acylators.

This divergence, which led to the specialization of the arylalkylamine N- acetyltransferases, was found to likely be a result of organization of the amine binding pocket, resulting in an active site less accommodating to long-chain acyl-CoAs.

The combination of kinetic analysis and crystallography, alongside phylogenetic analysis, shines light on the heterogeneity of insect AANATs and garners further

x discussion into their core differences, and some approaches possible to utilize these enzymes in insecticide design.

CHAPTER ONE:

INTRODUCTION AND MOTIVATION

Note to Reader: Portions of this chapter have been previously published in Biochemistry

& Molecular Biology Journal, 2018, 4, 1, 107-116 and has been reproduced as permitted by Insight Medical Publishing (See Appendix A).

1.1 Enzyme heterogeneity

The epigraph for this work is from Coco Chanel, the celebrated French fashion designer. Likely, her words “in order to be irreplaceable one must always be different” were in reference to her significant influence in the fashion industry, especially concerning the liberation of women from the suffocating clothing standards of the early 20th century.1

However, the quote rings true for many aspects of life, including in nature, as it effectively captures the essence of enzyme evolution and of enzyme heterogeneity. To be of enough use to a cell, i.e. to be worth the cost of transcription and translation, an enzyme must provide some contribution to the overall fitness of the organism.2 Any divergence away from the genetic formula of an enzyme will either be (and most often is) forgotten as a failed mutation, or accepted, potentially heralding the way to a more robust, more catalytically effective, or more promiscuous version of the original enzyme.3,4 This central dogma of evolution has meant that today, enzymes, having gone through millions of years of evolutionary stress and permutations, are highly ordered machines (Figure 1.1).

Everything about them has been tailored, from expression levels to location in the body, to suit the organisms harboring them.5

Figure 1.1: The hypothesized process of enzyme evolution. Divergence from a common ancestor leads to development of promiscuity and/or specialization, however the core structure maintains resemblance to ancestors and other family members

Enzyme heterogeneity from organism to organism is a fascinating and vital topic of research that has allowed us to understand our own evolutionary pathway, as well as that of other organisms. It has also allowed us to control the natural environment around

2 us to unprecedented levels through proteome manipulation. One key facet of this is in the advancement of genetic modification.6,7 For example, plants have evolved to utilize a cell wall for protection and structure.8 The polysaccharide involved in the rigidity of the cell wall, pectin, is broken down by the enzyme polygalacturonase.9–11 With some exceptions,12,13 animals have mostly evolved beyond the need for polygalacturonase,14 likely due to our incorporation of gut microbes which perform its role.15 In 1992, Calgene developed a form of tomato that was lacking in polygalacturonase. By removing the fruit’s own ability to break down pectin, a strain of tomato was produced, named the Flavr Savr tomato, which was made more resistant to rotting by slowing down pectin degradation.16

1.2 A brief history of pesticides

It is also by exploiting enzyme heterogeneity that we can chemically protect our crops, stored produce and livestock, and reduce insect-transmitted diseases. The basis of pesticide application is our ability to introduce a chemical, or group of chemicals, to the organisms we are protecting. This chemical has been specifically designed to interfere with pests, often through neurotoxicity, but minimize harm to non-targeted species, even if the target enzyme or pathway is present in a wide berth of organisms. The first recorded use of pesticides was by the Sumerians about 4,500 years ago, who applied sulfur compounds against insects.17 Many of the first noted uses of pesticides were natural products, such as pyrethrum, derived from the Chrysanthemum flower,18 and are still found in circulation today. However, by the 1940’s, and the expansion of the increasingly influential FDA, their relatively low selectivity and high levels of necessary application meant that alternatives needed to be developed. A new collection of inexpensive, potent synthetic pesticides was introduced to the market, led by dichlorodiphenyltrichloroethane

(DDT), a disruptor of insect sodium ion channels.19 The low toxicity DDT showed against mammals, and targeted use to reduce insect-borne diseases, led its pioneer, Dr. Paul

Muller, to the Nobel Prize in Physiology or Medicine in 1949.20 This introduced a period of contentment and, lamentably, an essence of naiveté, whereby with the apparent success of this new class of pesticides, the long-term effects of such chemicals were not in discussion. The release of Silent Spring in 1962 raised public awareness to the harms of DDT.21 The chemical was found to bio-accumulate in fatty tissues, with continued exposure leading to levels far higher than was considered ‘safe’. Among other concerns,

DDT was attributed to the decimation of the bald eagle population.22

The pesticide industry continued to grow in earnest, with many more synthetic pesticides becoming commonplace. These designer pesticides, such as neonicotinoids, introduced the industry to the next generation of pest control, with no bio- accumulation.23,24 Neonics, as neonicotinoids are often abbreviated, function as inhibitors of nicotinic acetylcholine receptors and are currently the most widely used insecticide.25

However, recent honeybee health problems, especially colony collapse disorder, have been attributed to the use (or claimed over-use) of many of these chemicals.26–30 Neonics became the focus of a disproportionate amount of research emphasizing the dangers of overuse. The rapid influx of research on the long-term effects of neonics was much welcomed,31 especially considering the mistakes made previously with DDT. The use of chronic sub-lethal toxicity to define the harm of neonics, however, does not give full credence to the relative toxicity and efficacy when compared to other insecticides.32,33

While a comprehensive assessment by Dively, et al. demonstrated that long-term exposure to neonics could have some marginal effects on queen numbers and

4 overwintering colony survival,34 many argue that neonics were merely on the wrong end of a witch hunt. Indeed, the neonicotinoid controversy arguably took attention away from other serious concerns. Due to the fact many pesticides on the market operate via a single mode of action to increase selectivity,35 the issue of pesticide resistance is one of the largest challenges faced by the industry going forward.

1.3 Insecticide resistance

It is apparent that through our attempts to control the environment, we are inadvertently acting as a driving force for evolution and change.36–38 Specifically, by introducing chemicals that target a particular metabolic pathway in an organism, we are putting that organism under enormous unnatural evolutionary strain to survive in its environment. Continued application of such an insecticide, for example, over several generations of the target insect means that those insects born with a resistant allele will be favored and carry on their resistance to proceeding generations (Figure 1.2).39–41 Since some level of resistance has been seen for almost every major insecticide,42 they are of primary concern, and are of most relevance to this body of text. Newly developed insecticides are quickly becoming classified as “non-renewable resources”, with a measure of effectiveness based on how long they will be commercially viable as a primary concern.

Figure 1.2: Proposed mechanism of pesticide resistance. As the organism is exposed to the pesticide, survivors carrying a resistant allele will be favored to pass on this resistance to future generations. (From Campbell Biology: Concepts & Connections, 7th, ©2012. Reprinted by permission of Pearson Education, Inc., New York, New York.

Resistance management has become a key component in the development of new insecticides, with the overall goal being to reduce the selection pressure for resistant genes, while not sacrificing the effect of the insecticide. To accomplish this goal, the World

Health Organization and the Food and Agriculture Organization of the United Nations released in 2010 the International Code of Conduct on the Distribution and Use of

Pesticides.42 The strategies outlined involve utilization of mixtures of insecticides with different modes of action, rotation or alternation of insecticides, treatment number restriction, and protection of beneficial organisms. The overarching tactic to combat

6 resistance, however, is through education; “it is important to learn as much as possible about the biology of the pest.”

1.4 AANATs as a potential insecticide target

This dissertation concerns itself with the development of knowledge on one particular class of enzymes which have been identified as potential insecticide targets; the arylalkylamine N-acetyltransferases. Arylalkylamine N-acetyltransferases (AANAT), often called dopamine- or serotonin N-acetyltransferases, are members of the Gcn5- related N-acetyltransferase (GNAT) superfamily of enzymes. These enzymes catalyze the acetyl-CoA-dependent acetylation of an amine or arylalkylamine (Figure 1.3).43

Figure 1.3: Mechanism for the AANAT-catalyzed N-acetylation of amines and arylalkylamines

Since the initial characterization of Drosophila melanogaster AANAT by Maranda et. al,44 a large number of insect genes encoding AANAT and AANAT-like proteins have been described, highlighting the biological importance of these enzymes.45–52 In vertebrates, AANAT catalyzes the rate-limiting step in the biosynthesis of melatonin and has a key role in regulation of the sleep-wake cycle.53–55 AANATs found in insects

(iAANATs) are similarly involved in important biological processes, including insect- specific roles. It is a testament to the heterogeneity of this group of enzymes that each

7 insect family has developed family-unique AANATs to facilitate their own needs. iAANATs are vital to cuticle morphology because they catalyze the acetylation of dopamine to N- acetyldopamine, a precursor to sclerotization, and hardening of the exoskeleton in many insects.47,52,56 In addition to dopamine, a variety of biogenic amine neurotransmitters also serve as acetyl group acceptors, meaning that the iAANATs are involved in neurotransmitter inactivation and, thus, may contribute to the regulation of neural signaling networks.57,58

An iAANAT-targeted insecticide would not only disrupt neural signaling, but would also inhibit cuticle development, decrease the structural stability, resulting in a non- conforming appearance (harming the insects’ ability to mate), and diminish the cuticle- mediated protection against injury and infection. The aforementioned AANAT-regulated production of melatonin (by catalyzing the acetylation of serotonin) manages the life span of Drosophila melanogaster, illustrating the toxicity potential of these biogenic amines if their inactivation is interrupted. Insects are particularly susceptible to iAANAT inhibition because insects lack the enzyme monoamine oxidase, an enzyme which functions in mammals to inactivate such biogenic.58 It has long been an endeavour of ours to examine the variety of functions of the iAANATs, and to ascertain the fatty acid amides they produce. Thus far, we have successfully identified, cloned and characterized iAANATs from Drosophila melanogaster and Bombyx mori.48–51,59–61 Other groups have characterized iAANATs from other insects, including Aedes aegypti, Tribolium castaneum and Antheraea pernyi.47,52,62,63 A picture of diversity and specificity for iAANATs emerges from the kinetic analyses showing a broad range of amine and acyl-CoA thioester substrates for the iAANATs, knockdowns demonstrating the detrimental effects of

8 targeting iAANATs, and sequence comparisons between the iAANATs revealing insect- specific targeting potential. All of these data point towards the iAANATs as excellent targets for the design of novel insecticides.

As amine specificity varies between iAANATs, so too does acyl-CoA chain length, ranging from acetyl-CoA to oleoyl-CoA. Based on the specificity of the acyl-CoA thioester substrates, some iAANATs function in the formation of both short- and long-chain N- acylamides, whereas others function to catalyze the formation of predominantly short- chain or predominantly long-chain N-acylamides. For example, iAANATs found in D. melanogaster show wildly differing results, especially with regards to acyl-chain length for their respective acyl-CoA substrates. Acetyl-CoA is a substrate for D. melanogaster Dm-

AANATA, KM = 39 ± 12 μM, but apparently had no appreciable affinity for the long-chain acyl-CoA, arachidonoyl-CoA.48 Yet in the same organism, Dm-AANATL2 accepted both acetyl-CoA and arachidonoyl-CoA as substrates, with KM values of 6.1 ± 0.3 μM and 1.9

± 0.25 μM, respectively, illustrating the differences in between the iAANATs found in the same organism.50,59 This trend is similarly seen when comparing two iAANATs expressed

60,61 by B. mori. Acyl-chain length had negligible effect on the KM values for the acyl-CoA substrates for Bm-iAANAT while Bm-iAANATL3 preferred acyl-CoA substrates with an acyl chain length of 2-10 carbon atoms, with longer chain acyl-CoA thioesters not acceptable as substrates, at all (Table 1.1).

Table 1.1: KM values for the iAANATs found in D. melanogaster and B. mori with respect to different acyl chain lengths

(KM) (μM) Dm- Dm- Bm- Bm- Acyl-CoA AANATA48 AANATL250 iAANATL361 iAANAT60,64 Acetyl-CoA 39 ± 12 6.1 ± 0.27 90 ± 3.6 0.31 Butyryl-CoA 36 ± 2 1.8 ± 0.17 14 ± 0.75 N/A Hexanoyl-CoA 23 ± 3 N/A 13 ± 0.54 N/A Octanoyl-CoA 18 ± 3 N/A 10 ± 1.0 N/A Decanoyl-CoA 220 ± 60 N/A 12 ± 5.5 N/A Palmotyl-CoA N/A 9.9 ± 1.6 N/A 1.1 ± 0.3 Oleoyl-CoA N/A 3.6 ± 0.58 N/A 1.7 ± 0.51 Arachidonyl-CoA N/A 1.9 ± 0.25 N/A 1.2 ± 0.67

A review of the data for the amine substrates for the iAANATs again point to specific metabolic functions for the specific members of the iAANAT family. Differences of several orders of magnitude are found for amine KM values when comparing iAANATs

(Table 1.2). For example, the KM for dopamine with Dm-AANATA is 25 ± 2 μM and that for Bm-iAANATL3 is 330 ± 23 μM. Differences like this are congruent for numerous amines, signifying an evolutionary divergence in binding specificity in the active site between iAANATs.

Table 1.2: Binding constants for iAANATs found in D. melanogaster and B. mori with respect to different amine acceptors

(KM) (μM) Dm- Dm- Dm- Bm- Amine AANATA48 AANATL749 AgmNAT51 iAANATL361 Tyramine 12 ± 1 42 ± 2 N/A 63 ± 6.7 Octopamine 10 ± 2 120 ± 10 N/A 18 ± 0.77 Dopamine 25 ± 2 170 ± 20 N/A 330 ± 23 Tryptamine 33 ± 3 26 ± 2 N/A 97 ± 8.8 Norepinephrine 32 ± 6 230 ± 50 N/A 140 ± 8.6 Phenethylamine 56 ± 13 320 ± 40 N/A N/A Serotonin 110 ± 8 160 ± 20 N/A 1100 ± 63 4-methoxyphenethylamine 780 ± 60 190 ± 20 N/A N/A 4-phenylbutylamine 270 ± 20 610 ± 30 N/A N/A 3,4- N/A N/A 3200 ± 300 320 ± 30 dimethoxyphenethylamine Histamine N/A 520 ± 50 N/A 1700 ± 98 Putrescine N/A 81 ± 11 51 ± 3 N/A Agmatine N/A 790 ± 100 0.3 ± 0.02 N/A Spermine N/A N/A 18 ± 3 N/A N8-acetylspermine N/A N/A 9.1 ± 1 N/A Spermidine N/A N/A 17 ± 0.5 N/A Cadaverine N/A N/A 32 ± 6 N/A

Kinetic analyses provide a limited evaluation of the active site differences between the iAANATs. The KM values are not Kdissociation values and a comparison (or ratio) of KM values may only provide an estimate ratio of Kdissociation values for an acyl-CoA or amine substrate. However, the highlighted differences in the KM values imply a large enough distinction in substrate affinity to suggest that insecticides can be developed that will only target iAANATs in insect pests. Another aspect in considering the iAANATs as insecticide targets is to evaluate expression knockdown data. If the expression knockdown of a potential insecticide target is not deleterious to the insect, then an inhibitor targeting it will probably not be a useful insecticide. One such knockdown was performed by Long, et al. 11 on Bombyx mori iAANAT2.64 The inhibition of Bm-iAANAT2 expression led to an increase in melanin deposition in larvae and adults, resulting from increase cellular concentrations of dopamine. As mentioned, any deviation in the appearance of the insect would have a naturally detrimental effect on its chances of mating.65 Another iAANAT knockdown was performed in T. castaneum, the red flour beetle, an insect regarded as a serious pest to the food industry and hence a perfect model for insecticide target discovery.52 Noh et. al. demonstrated that along with darkening of the cuticle, the knockdown resulted in a separated, misshapen elytron (the wing casing of the beetle), and misfolding of the hindwings of the beetle. The evidence demonstrates that targeting iAANAT compromised the structural integrity of the T. castaneum exoskeleton. This naturally leaves the insect vulnerable to a host of environmental threats, including disease, predation, and the aforementioned inability to mate.

While the experimental data point toward the iAANATs as excellent targets for insecticide development, insecticides, as mentioned, have come under scrutiny for their toxicity against the honeybee, Apis melifera, and the bumblebee, Bombus terrestris. It is here the iAANATs stand out as insecticide targets. An examination of the sequences for numerous iAANATs reveals several unique iAANAT families. The “traditional” iAANAT gene is easily identifiable through a CoA binding pocket motif (synonymous with the NAT superfamily) and an insect specific motif, FxDEPLN. This motif contains functionally and structurally important residues. Due to the link of the enzyme to dopamine acetylation, this “traditional” iAANAT is often referred to as dopamine N-acetyltransferase (DAT).44

DAT is conserved among all insects. However, genome mining suggests that most insects contain several other types of iAANATs, also with characteristic motifs. These

12 other families of iAANATs generally exhibit very low sequence homology outside of their phylogenetic grouping. Generally speaking, there is less than 30% homology between

AANATs from insect to insect; and even between iAANAT families within the same species, this number is usually around 40%. To add to this, A. melifera and B. terrestris both contain an iAANAT specific for Apidae (bees). Coleoptera (beetles) contain several more. This is further illustrated in Figure 1.4.

Figure 1.4: Primary sequence alignment of various insect AANATs, as well as human serotonin N-acetyltransferase (SNAT). DAT specifies the conserved dopamine N- acetyltransferases found in all insects. iAANATs are the genus specific AANATs. Highlighted in green is the amine binding pocket and highlighted in red are residues that make up the CoA binding pocket. Both are noticeably generally conserved among DATs but varied among genus-specific iAANATs. The nomenclature and associated GenBank accession codes are as follows: AA – Aedes aegypti; DM – Drosophila melanogaster; TC – Tribolium castaneum; BT – Bombus terrestris; AM – Apis mellifera; HS – Homo sapiens. ‘AA DAT’ – XP_001661400.1; ‘DM DAT’ – NP_995934.1; ‘TC DAT’ – XP_972873.2; ‘BT DAT’ – XP_012167469.1; ‘AM DAT’ – XP_016770090.1; ‘BT iAANAT’ – XP_020723819.1; ‘AM iAANAT’ – XP_001122379.2; ‘AA iAANAT’ – XP_001649422.1; ‘TC iAANAT’ – XP_973841.1; ‘HS SNAT’ - NP_001079.1

Importantly, the regions responsible for binding of both the acyl-CoA and the amine substrate vary considerably between these iAANAT families, despite very high structural homology. In all iAANATs, the overall secondary and tertiary structure of the amine binding pocket is very similar. However, differences in specific residues are responsible for changes in binding affinities. In Diptera- (fly) specific iAANATs, for example, many hydrophobic residues are replaced with residues containing nucleophilic groups and hydrogen bond acceptors that are not found in other iAANATs.57 This suggests that by exploiting these catalytically relevant, yet insect-specific residues, it is possible to design compounds that bind to a specific iAANAT family, such as Diptera iAANAT or Coleoptera iAANAT, which would not bind (or would bind with low affinity) to others, leaving non- targeted species unaffected.

These comparisons of iAANATs reveal the potential for high insecticide specificity, by solely targeting pests or disease vectors, and keeping agriculturally or ecologically important species unharmed. Additionally, iAANATs share little to no sequential homology to AANATs expressed in vertebrates. This is in stark contrast to the targets of many currently used insecticides, such as the aforementioned neonicotinoids, as well as organophosphates and organochlorines, which often have issues with non-specific targeting,66 and often cause severe environmental side-effects.67 Further work must be done to design and develop iAANAT-specific inhibitors to solidify these as legitimate targets. However, the evidence discussed herein indicates the iAANATs are excellent insecticide targets and the development of iAANAT-targeted inhibitors has the potential to solve many of the problems faced by current insecticide development.

The overarching goal of this dissertation is to develop our understanding of iAANATs both structurally and kinetically. Chapter 2 outlines much of the preceding research with regards the kinetic and chemical mechanism of iAANATs and their biological role in insects. Chapter 3 is a case study on the heterogeneity of iAANATs, with

AANAT1 from T. castaneum as the subject. Chapter 4 presents a thorough characterization of a novel AANAT from T. castaneum, named TcAANAT0. The kinetic and structural information gained from this enzyme will be invaluable in the presentation of iAANATs as a viable insecticide target. Chapter 5 details the mechanistic characterization of Bm-iAANAT3 from Bombyx mori, one of the first AANATs reported with a random sequential mechanism. Finally, Chapter 6 addresses a long-standing question concerning AANATs; their role in long-chain fatty acid amide production. It is hoped that the discussions presented herein establish the significance of iAANAT heterogeneity and its place as a next generation insecticide target.

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CHAPTER TWO:

INSECT AANATS: MECHANISMS AND ROLE IN FATTY ACID AMIDE

BIOSYNTHESIS

Note to Reader: Portions of this chapter has been previously published in Frontiers in

Molecular Biosciences, 2018, 5, 66 and has been reproduced as permitted by Frontiers

(See Appendix A).

2.1 Introduction

The N-acylation of aromatic monoamines is mostly associated with the acetylation of serotonin to form N-acetylserotonin, an N-acylarylalkylamide precursor in the formation of melatonin.1,2 Production of N-acetylserotonin is the rate-determining step in the biosynthesis of melatonin and the enzyme responsible is arylalkylamine N- acyltransferase (AANAT). The rhythmic production of melatonin, which regulates circadian rhythms in mammals, correlates to rhythmic changes in AANAT activity.3,4 For this reason, AANAT has been labeled the “timezyme”.5 Thus far, only one AANAT has been characterized in mammals and most other vertebrates, with the exception being bony fish, which contain an assortment of three AANAT isoforms.6,7 However, it has been suggested that other AANAT-like enzymes are likely to exist in mammals because while

AANAT is primarily expressed with paracrine roles in the pineal gland and eyes, N- acylaryalkylamides are found in several regions of the body.2,8,9

In contrast to vertebrates, non-vertebrates, including insects, express multiple

AANATs in order to regulate aromatic amino metabolism.10,11 For example, at least thirteen and eight putative iAANATs have been identified in Aedes aegypti and Drosophila melanogaster respectively.12–14 There are a number of plausible reasons why insects express multiple iAANATs. Firstly, monoamine oxidase (MAO) activity is limited in insects.15 In mammals, MAO is critical to the inactivation of catecholamines and other aromatic amines.16 The inability to catabolize the catecholamines is linked to schizophrenia, apnoea, psychosis, as well as physiological disorders related to prolactin inhibition.17,18 To avoid a toxic buildup of these constituents, insects recruit numerous iAANATs to inactivate catecholamines by N-acetylation.11,19 Secondly, the product of the iAANAT-catalyzed acetylation of dopamine, N-acetyldopamine, is a key component in the cuticle sclerotization process for many insects.20,21 Knockdown of AANAT associated with dopamine N-acetylation in Bombyx mori, and Tribolium castaneum resulted in an increase in intracellular concentrations of dopamine and other biogenic alkylamines and the overproduction and deposition of melanin.22–24 The lack of acetylated dopamine gave rise to abnormalities in the wing casings, misfolding of the hind wings, and a darkened and malformed exoskeleton due to melanin overproduction. The cuticle is a vital barrier from the environment, protecting the insect against injury and infection, while also providing structural stability.25,26 Knockdown of Drosophila melanogaster AANATL2, associated with the formation of long-chain N-acyldopamines and N-acylserotonins resulted in decreased levels of N‐palmitoyldopamine, a proposed regulator of the

Hedgehog signaling pathway.27,28

The low sequence homology (usually 20 – 40% identity) of these enzymes from insect to insect implies that iAANATs could be a viable target for novel insecticide design.29,30 Additionally, phylogenetic analysis demonstrates several apparent sub- groups of iAANAT that could potentially offer specific targeting. These sub-groups are best defined based on their substrate specificity. For example, a neighbor-joining tree

(Figure 2.1) of all characterized iAANATs divides these enzymes (with some exceptions) into those which demonstrate standard dopamine-N-acetyltransferase activity, polyamine

N-acetyltransferase activity, and a more insect-specific N-acetyltransferase activity.31–34

The functions of these sub-groups are delegated among cuticle sclerotization, neurotransmitter activation, and long-chain fatty acid amide formation, with some iAANATs likely covering multiple roles.

It is apparent much remains to be unearthed about the iAANATs and their role in catecholamine and aromatic amino acid metabolism. Much of this work comes from examining the enzymes directly, for clues on how and why they function kinetically and chemically. Presented in this chapter is an in-depth analysis of the structural and functional relationships of the iAANATs and how the iAANATs contribute to the N- acylation reactions of aromatic amino acid metabolism in insects. A recent review has been published on iAANATs, albeit with a different focus than that outlined here.11 It is expected that this current chapter coupled to the review of Hiragaki and colleagues provides a thorough analysis of the current state of knowledge of the iAANATs.

Figure 2.1: Neighbor-joining tree built using Poisson-corrected distances on characterized insect arylalkylamine N-acetyltransferases (iAANATs). The orange box represents probable typical insect dopamine-N-acetyltransferases. The blue box represents probable polyamine N-acetyltransferases. The red box represents putative insect-specific N-acyltransferases. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA7.

2.2 N-Acetylation of the aromatic acids and aromatic amino acid – derived

metabolites

Early work identified the aromatic amino acids and other catecholamines in insects.21,25,35,36 With the exception of tyrosine, insects are incapable of the de novo synthesis of the aromatic amino acids and, thus, histidine, phenylalanine, and tryptophan

29 are either obtained from the diet or via a symbiotic relationship with bacteria, fungi, and/or plants.37–40

Histidine, phenylalanine, tyrosine, and tryptophan serve as the precursors to other bioactive compounds in insects, including other aromatic amino acids that are not on the canonical list of the 20 typically found in proteins: 3,4-dihydroxyphenylalanine (DOPA), dopaquinone, kynurenine, and xanthommatin. The focus of this chapter is on the enzymatic acylation of the α-amino group of the aromatic amino acids, which stems from our interests in the biosynthesis of the fatty acid amides. Other chemical transformations of specific aromatic amino acids in insects are discussed elsewhere: oxidation,41,42 decarboxylation,43,44 hydroxylation,45,46 transamination,47,48 phosphorylation,49,50 halogenation,51,52 and sulfation.53

Enzymes that catalyze the acyl-CoA-dependent acylation of the α-amino group of aromatic amino acids are members of the Gcn5-related N-acetyltransferase (GNAT) superfamily of enzymes.54,55 They have been described from insects under a variety of names, including dopamine N-acetyltransferase, indolamine N-acetyltransferase, indoleamine N-acetyltransferase, serotonin N-acetyltransferase, spermidine N- acetyltransferase, agmatine N-acetyltransferase, and arylalkylamine N-acetyltransferase

(AANAT).11 Much of the focus on these enzymes has been on amine N-acetylation, with acetyl-CoA serving as the acetyl group donor. This is because of the importance of dopamine acetylation in the sclerotization process,20 of serotonin acetylation in melatonin biosynthesis,1 and of biogenic amine acetylation in their inactivation.42 It is also notable that N-acetyltyrosine and N-acetylhistidine have been reported as metabolites in insects.56,57

Figure 2.2 The reaction catalyzed by arylalkylamine N-acyltransferase.

Fatty acid amides represent a large family of biologically occurring lipids of the

58,59 general structure, R-CO-NH-R1. This structural simplicity belies a wealth of diversity amongst this lipid family as the R-group is derived from fatty acids (R-COOH) and the R1- group is derived from the biogenic amines (H2N-R1). N-Fatty acyl derivatives of histidine, phenylalanine, tyrosine, and tryptophan have been identified in insects.60,61 In addition,

N-fatty acyl derivatives of dopamine and serotonin have been identified in D. melanogaster.62,63 We have proposed that novel iAANATs exist which will catalyze the acyl-CoA-dependent formation of these N-fatty acylamides (Figure 2.2). AANATs in D. melanogaster and B. mori have been identified that will utilize long-chain fatty acyl-CoA thioesters as substrates leading to the production of these fatty acylamides.62,64 Thus, we suggest replacing the name “N-acetyltransferase” with “N-acyltransferase” to better reflect the most current data on this family of enzymes. Note that all the iAANATs characterized to date will accept aromatic amino acid-derived metabolites as substrates

(dopamine, serotonin, tyramine, tryptamine, phenethylamine, and/or octopamine), but none of the iAANATs that have been characterized will accept the aromatic amino acids as substrates.14,30,62,65–70 In fact, we have reported that the aromatic amino acids and tyrosine methyl ester do not bind to three D. melanogaster AANATs, DmAANATA,

DmAANATL2, and DmAANATL7, with any appreciable affinity (Kd > 10 mM) because these compounds show no inhibition at 1.0 mM. 14,62,66 31

The AANAT-catalyzed formation of N-acylamides is not the only reaction thought to account for the synthesis of these molecules in insects. While thermodynamically unfavorable under biological conditions, the biosynthesis of N-linolenoyl-L-glutamine in

Manduca sexta has been attributed to the direct conjugation of unactivated linolenic acid to L-glutamine.71

N-β-Alanyldopamine (NBAD) and N-β-alanylhistamine (carcinine) are two other N- acylated aromatic amino acid-related compounds found in insects.72,73 These are produced by an ATP-dependent reaction between β-alanine and dopamine or histamine, catalyzed by the enzyme NBAD synthase (also known as Ebony). Mechanistic studies of

NBAD synthase show that β-alanine is initially activated by a reaction with ATP to yield

β-alanyl-AMP and pyrophosphate.74,75 The β-alanyl moiety is then transferred to the sulfhydryl group of 4’-phosphopantetheine, a prosthetic group attached to Ser-611 in the

D. melanogaster enzyme. Nucleophilic attack by the amino group of dopamine or histamine at β-alanyl-thioester yields NBAD or carcinine (Figure 2.3).

Figure 2.3: The reaction catalyzed by NBAD synthase. The structures of two products of the NBAD synthase reaction, NBAD and carcinine, are in the inset

2.3 Insect AANAT – Kinetic mechanism

Due to the bi-substrate, bi-product (bi-bi) nature of the reaction, there are two possibilities for the iAANAT kinetic mechanism: sequential or non-sequential (ping-pong).

A sequential kinetic mechanism is divided further into either ordered or random mechanisms by determining if one of the substrates requires the formation of an enzyme- substrate (E•S) complex before binding.76 Protocols to differentiate between steady-state ordered and rapid-equilibrium ordered bi-bi kinetic mechanisms involve initial velocity kinetic experiments varying one substrate at different fixed concentrations of the second substrate in the presence and absence of a dead-end inhibitor. The trends given by these types of experiments are fingerprints for the elucidation of the kinetic mechanism.

Supplementary experiments such as measurements of direct substrate binding and/or kinetic isotope effects are often illustrative. Both sequential and ping-pong mechanisms have been attributed to iAANATs; however, the most common is a sequential mechanism.

Early studies of an iAANAT from the American cockroach, Periplaneta americana. demonstrated that this enzyme functioned via a sequential kinetic mechanism.68,77 iAANATs from other insects were purified and characterized following the work on the P. americana AANAT. iAANATs from Acyrthosiphon pisum (pea aphid),78 Aedes aegypti

(yellow fever mosquito),12,69 Antheraea pernyi (silkmoth),30 Bombyx mori (silkworm),22,70

Dianemobius nigrofasciatus, (band-legged cricket),79 Drosophila melanogaster (fruit fly),13,80,81 and Tribolium castaneum (red flour beetle),24 were purified and characterized, often including a set of biogenic amine substrates evaluated using acetyl-CoA as the acyl donor. However, determination of the kinetic mechanism for these various iAANATs was not included in these cited works.

Our work in this field started with the hypothesis that iAANATs were responsible, not just for short chain N-acylation, but also for long chain N-acylation to form N- oleoylated, N-palmitoylated, and, perhaps, N-arachidonylated amines. Drosophila melanogaster presented the ideal model organism; the flies were known to produce N- fatty acylamides and to express at least eight different iAANATs.13,61,63 Dempsey et al. demonstrated that D. melanogaster AANATA (DmAANATA) followed an ordered sequential mechanism, with acetyl-CoA binding first and catalysis only taking place after the formation of the DmAANATA•acetyl-CoA•tyramine complex.14 We built on our work on DmAANATA leading to expression and characterization of three other AANAT-like enzymes from D. melanogaster, DmAANATL2,62 DmAANATL7,66 and DmAANATL8.65

The best substrates for DmAANATL8 were agmatine and acetyl-CoA, so we renamed

DmAANATL8 as DmAgmNAT – agmatine N-acetyltransferase. Our data for all three of these D. melanogaster iAANATs were consistent with a steady-state ordered kinetic mechanism with the acyl-CoA substrate binding first for all three of these DmAANATs.

We have recently broadened our scope beyond D. melanogaster to Bombyx mori, another insect known to express multiple iAANATs. Three B. mori iAANATs have been described and partially characterized: Bm-iAANAT,64,70 Bm-iAANAT2,22 and, described in

Chapter 5, Bm-iAANAT3.82 We found that the kinetic mechanism for Bm-iAANAT3 was steady-state ordered with the acyl-CoA substrate binding first, similar to kinetic mechanisms elucidated for iAANATs from D. melanogaster and P. americana. Structural studies by Aboalroub et al. revealed that the binding of acetyl-CoA to Bm-iAANAT3 alters the conformation of the enzyme to facilitate binding of the amine substrate.83 In the

35 absence of acetyl-CoA, the amine substrate binds with relatively low affinity to Bm- iAANAT3.

2.4 Insect AANAT – Catalytic mechanism

The kinetic mechanism of an enzyme provides only a partial insight of the intricacies of enzymatic catalysis. To get a more complete picture of the specific interactions that mediate catalysis, work must be done to elucidate a chemical mechanism. Delicate balances between pH, residue positions, and both local and long- ranged readjustments in active site orientation offer a fascinating conundrum which is only now beginning to be understood thanks to a combination of experimental, computational and quantum mechanical studies. To investigate the catalytic mechanism is to understand the very intricate subtleties that regulate enzyme catalysis.

One goal of the mechanistic work on the iAANATs is to understand the pH- dependence of catalysis regarding the protonation state of critical active site amino acids.

Asano and Takeda first noted the importance of pH to iAANAT activity in their work on the P. americana iAANATs.77 They identified one acidic and one basic form of the P. americana enzymes. The optimal pH for acidic iAANAT was approximately pH 6.0, whereas that for the basic form was approximately pH 10. Definitive mechanistic conclusions about the P. americana iAANATs could not be drawn because their data was generated using only one concentration of both substrates rather than a more complete pH-rate profile obtained by varying both substrates as pH was varied. To date, only one other iAANAT, from D. nigrodasciatus has been described that has maximum activity at pH < 7.0.79

Activity profiles as a function of pH were reported for two iAANATs from A. aegypti,

AaNAT2 and AaNAT5b. Activity was maximum between pH 8 and 9 with either a decrease in activity as pH was increased (AaNAT5b) or a relatively constant level of activity as pH was increased (AaNAT2).12 Similar trends were noted in the pH vs. activity data for other iAANATs in Antheraea pernyi and Tribolium castaneum, except that the maximum activity was observed at a lower pH, pH 7.5 to 8.5.24,30

While these data are interesting and useful, these studies do not provide conclusive mechanistic information about iAANAT-mediated catalysis. Instead, a more complete analysis (varying both substrates) to measure the pH-dependence of the KM, kcat, and kcat/KM values must be undertaken. Such studies were carried out on four iAANATs from D. melanogaster, DmAANATA.14,80 DmAANAT2,62 DmAANATL7,66 and

DmAgmNAT.65

Some overlapping trends were observed in the pH-rate profile data on these four

D. melanogaster AANATs. Catalysis was dependent on a general base, which exhibited a pKa of approximately pH 7.5. A similar trend was observed in pH rate profiles of Bm-

82 iAANAT3, except the general base had a pKa of approximately 6.8 for this enzyme. One other interesting nuance of the pH rate data on the D. melanogaster and B. mori AANATs was a bell-shaped curve, indicating the presence of a second catalytically important

14,67,80 residue with a pKa of 9-10. One suggestion to account for the second pKa is an active site Ser serving as a general acid to protonate the thiolate anion of coenzyme A

(CoA-S-) (Cheng et al., 2012).80 An active site Ser serving as a general acid is unusual in enzymatic chemistry because this would require a significant decrease in pKa of the serine hydroxyl from 13-14 to 9-10. More likely, this second pKa reflects the pKa of the departing

84 - CoA-SH, pKa = 9.6 to 10.4, meaning that that the release of CoA-S is slower for the iAANATs.

Alanine-scanning mutagenesis, sequence alignments, and crystallographic data

(when available) were all use to alleviate the ambiguity of some of the results and to assign residue-specific roles in iAANAT catalysis. Sequence alignments demonstrated several conserved motifs, representative of structurally or catalytically relevant residues

(Figure 2.4). Within the iAANATs, we noted a highly conserved DEPLN motif (Figure 2.5)

– an obvious target for mutagenesis. Mutation of the glutamate in this motif to alanine,

Glu-47 in DmAANATA,14 Glu-26 in DmAANATL7,66 Glu-34 in DmAgmNAT,65 and Glu-27

Bm-iAANAT3,82 resulted in an almost complete eradication of iAANAT activity. The pH- rate profiles for Glu-to-Ala mutants in DmAANATA, DmAANATL7, and DmAgmNAT strongly supported a role for the Glu in the DEPLN motif serving as the general base during catalysis. For the mutant iAANATs, the acidic pKa disappeared and the resulting pH-rate profiles were either flat (no dependence of the residual rate on pH) or exhibited a linear relationship with pH (slopes from 0.2 to 0.7) suggesting a “rescue” of catalytic activity by hydroxide as the pH increased. The mutagenesis data also show the Glu in

DEPLN motif must function in substrate binding since the respective Glu-to-Ala mutants.

We must point out that the Glu in the DEPLN also has a role in amine binding because the KM,amine values increased significantly for the E47A mutant in DmAANATA, the E26A mutant in DmAANATL7, and the E34A mutant in DmAgmNAT.

Figure 2.4: Sequence alignment of iAANATs. Residues are colored depending on acidic (red), basic (blue), polar (green), or hydrophobic (yellow). Cysteines are in gold and glycine is purple. Aedes aegypti – AaNAT1 (GenBank accession no. XP_001661400); AaNAT2 (GenBank accession no. XP_001663122); AaNAT5b (GenBank accession no. XP_001649916); Bombyx mori – Bm-iAANAT (GenBank accession no. NM_001079654.2); Bm-iAANATL3 (GenBank accession no. NM_001190842.1); Drosophila melanogaster – DmAANATA (GenBank accession no. NM_079115.3); DmAANATB (GenBank accession no. NM_206212.1); DmAANATL2 (GenBank accession no. NM_135161.3); DmAANATL7 (GenBank accession no. NM_130653.3); DmAgmNAT (GenBank accession no. NP_572268.1); Periplaneta americana – PaNAT (GenBank accession no. BAC87874.1); TcAANAT1 – Tribolium castaneum (GenBank accession no. NM_001145908.1)

Figure 2.5: Crystal structure of DmAANATA (PDB: 3TE4) active site with bound acetyl- CoA. Residues shown in cyan represent DEPLN region conserved among many iAANATs

One exception to the pattern of results obtained for DmAANATA, DmAANAT7,

DmAgmNAT, and Bm-iAANAT3 was DmAANATL2.67 The E29A mutant for DmAANATL2 showed only a relatively slight decrease in the kcat relative to wildtype as well as a bell- shaped pH-rate profile yielding the same pKa values as wildtype. Thus, for DmAANATL7 the general base required for catalysis with a pKa value of ~7.4 is not Glu-29 and is currently unknown. As we found for the other D. melanogaster AANATs, the Glu in the

DEPLN motif for DmAANATL2 has a role in amine substrate binding because the KM,amine increased ~20-fold relative to wildtype without any change in the KM,acetyl-CoA value.

Availability of a crystal structure for DmAANATA (PDB accession code: 3TE4)80 and DmAgmNAT (PDB accession code: 5K9N)65 allowed for more in-depth structural analysis. Combined with this, homology models were developed using SWISS-MODEL for DmAANATL2 (based on 3TE4 and 5GIF) and DmAANATL7 (based on 4FD6).12,85

Both Glu-47 in DmAANATA and Glu-34 in DmAgmNAT are located in their respective

active sites with several structural waters positioned within proximity (Figure 2.6). This

led to the suggestion that these water molecules form a “proton wire” to assist the general

base in catalysis by facilitating proton transfer, as had been suggested in other GNAT

enzymes.54

Figure 2.6: Crystal structure of DmAANATA (PDB: 3TE4 - cyan) active site with bound acetyl-CoA aligned with apo structure of DmAgmNAT (PDB: 5K9N - orange). Highlighted residues indicate Glu-47 and Glu-34 which function respectively in these enzymes as the apparent general base. The surrounding water molecules (colored spheres) enable a “proton wire” to facilitate proton transfer.

Sequence alignment of the iAANATs also revealed a highly conserved arginine

residue, Arg-153, which based on the crystal structure of DmAANATA, seemed

necessary in maintaining structure. A salt bridge is formed between Arg-153 and Asp-46

80 (Figure 2.7). Surprisingly, the kcat for the R153A mutant in DmAANATA was ~5-fold

higher than wildtype. Corresponding increases in the KM for both substrates resulted in

14 the kcat/KM for the R153A being 2-5 fold lower than wildtype. We attributed the results for the R153A mutant to elimination of the R153-D46 salt bridge that is critical to a

DmAANATA conformation that decreases the rate of CoA-SH release. This argument suggests that Arg-153 does not have a direct role in catalysis and, further, points towards a partially rate-determining conformational change in DmAANATA, which has been observed in other GNAT enzymes.54,55 Arg-138 in DmAANATL7 and Arg-138 in

DmAANATL2 are equivalent to Arg-153 in DmAANATA. Data generated for the R138A mutant in DmAANATL7 was similar to what was found for the R153A mutant of

DmAANATA, again, arguing against a direct role of Arg-138 in DmAANATL7 catalysis

Figure 2.7: Crystal structure of DmAANATA (PDB: 3TE4 - cyan) active site with bound acetyl- CoA aligned with homology models of DmAANAT2 (magenta) and DmAANATL7 (yellow). Illustrated by the yellow dashes is the salt bridge that forms between Asp-46 and Arg-153 of DmAANATA.

42 and for a partially-rate determining conformation change regulating the release of CoA-

SH.66

DmAANATL2 proved different.66 Mutation of Arg-138 to Ala in DmAANATL2 resulted in kcat values that are ~20% of the wild-type enzyme and KM values similar to the wild-type. These data imply that Arg-138 may have a direct role in DmAANATL2 catalysis and that a conformational change involving Arg-138 is not particularly rate-determining for this iAANAT. Homology modeling of DmAANATL2 based on DmAANATA (PDB access code: 3TE4) indicates a conserved position for the respective arginine residues.

The different effects mutation of this residue has on kcat for both enzymes implies that

Arg-138 of DmAANATL2 may share catalytic and conformational responsibilities. An alternative residue perhaps fills the role for this catalytic residue in wild-type DmAANATA and in DmAANATL7, or possibly in the mutated species as a “rescue” in the absence of the arginine residue. HSQC-NMR titrations of both wild-type and mutated species could be employed to examine this phenomenon. However, because of the apparent aggregation of many of these enzymes at high concentrations, NMR is usually difficult and often impossible to perform.

The structure of DmAANATA indicated that His-220 was in van der Waals contact with Pro-48 of the active site (Figure 2.8A).80 Sequence and structural alignments of iAANATs reveal this His to be relatively well conserved (Figure 2.8B). The H220A mutant exhibited 4-7-fold increase in KM and a 4-fold decrease in kcat – a trend observed in the corresponding His-to-Ala mutants in DmAANATL7 (His-206) and in DmAgmNAT (His-

206). The DmAgmNAT crystal structure demonstrated clearly that His-206 was important to the formation of the active site through interaction with numerous residues in its

43 environment (Figure 2.8C). DmAANATL2 was again an outlier in this aspect, with mutation of its respective histidine, His-206, resulting in a mutant enzyme completely devoid of catalytic activity.66 The creation of an inactive H206A mutant of DmAANATL2 is difficult to interpret. His-206 may be essential to structural integrity of the enzyme or may have an essential role in catalysis.

Figure 2.8: The importance of the active site histidine of iAANATs in the structural maintenance of the active site. (A) Crystal structure of DmAANATA (PDB: 3TE4 - cyan) active site with bound acetyl CoA. Spheres indicate relative proximity of His-220 and Pro- 48, which in turn facilitates van der Waals contact. (B) Alignment of DmAANATA (PDB: 3TE4 - cyan), DmAgmNAT (PDB: 5K9N - orange), DmAANAT2 (magenta) and DmAANATL7 (yellow) demonstrating conservation of this active-site histidine. (C) Crystal structure of DmAgmNAT (PDB: 5K9N - orange) highlighting numerous interactions of His- 206 with surrounding residues, confirming its importance in active-site formation.

While one set mechanism has not been agreed upon for iAANAT catalysis, there are at least two plausible suggestions that agree with the available data. The first

44 represents an ordered sequential mechanism, where the acyl-CoA binds first, followed by the amine substrate. This leads to the formation of an iAANAT•acyl-CoA•amine ternary complex before catalysis can occur. From here, a catalytic base (generally a glutamate, but possibly a histidine in DmAANATL2) deprotonates the positively charged amine moiety through the use of a “proton wire” of ordered water molecules. Nucleophilic attack of the carbonyl of the acyl-CoA thioester generates a zwitterionic tetrahedral intermediate.

This collapses as the CoA-S- is protonated by the positively charged amine of the intermediate, thus, relinquishing the two products, most likely in the order of N-acylamide first, followed by CoA-SH (Figure 2.9). This mechanism is equivalent to that proposed for serotonin N-acetyltransferase found in mammals.86

The second mechanism proposed represents the involvement of a general acid.

Following binding of the substrates to form the iAANAT•acyl-CoA•amine ternary complex, nucleophilic attack of the carbonyl of the acyl-CoA thioester by the amine generates a zwitterionic tetrahedral intermediate. Collapse of this is catalyzed by a general base deprotonation of the positively charged amine intermediate, as well as a general acid protonation of CoA-S-, yielding the two products. While this mechanism cannot be eliminated by the available mechanistic data on the iAANATs, this mechanism is less favored, due to questions about an active site amino acid serving as a general acid during catalysis.

Figure 2.9: Proposed catalytic mechanisms for iAANAT. (A) Mechanism demonstrating a general base functioning to deprotonate the positively charged amine yielding a zwitterionic tetrahedral intermediate. Subsequent protonation and release of the CoA-SH product yields the final fatty acylamide product (B) Mechanism demonstrating both a general base and general acid functioning in intermediate collapse and release of the CoA-SH and fatty acylamide products

2.5 Insect AANAT – a “timezyme”?

As mentioned, in vertebrates, AANAT is involved in regulating circadian rhythms.

It cannot be assumed, however, that the AANATs function in the same way in insects.

Circadian rhythms are any biological process that follow a daily cycle; sleeping at night and being awake in the day is a common example of a light-related circadian rhythm. The driving force behind the circadian rhythm of any organism is the innate biological clock.

The rhythmic pattern of this clock is maintained through a complex, feedback induced, pathway associated with clock genes and their related proteins.87 Entrainment of this pathway is controlled by mediation of melatonin levels,88 which is dependent on photoperiodic messages, i.e. light exposure to the eyes. The changes in melatonin levels lead sequentially to photoperiodic responses. Vertebrate AANAT is expressed in photosensitive organs such as the pineal gland, retina, and parietal eyes demonstrating a clear association with photoperiodic signaling in vertebrates;11,89 thus, AANAT was termed the ‘timezyme’.5 In insects, it is unknown how photoperiodic signaling is integrated into daily rhythms because insect eyes are usually insufficiently photosensitive.3,90 This suggests insects may rely on other environmental cues rather than light alone to distinguish between night and day. A few insects, however, have shown the ability to interpret photoperiodic stimuli, namely P. americana, L. migratoria, D. nigrofasciatus, and

L. hedyloidea.79,91–93

The classification of an iAANAT as a ‘timezyme’ is unclear because the exact role played by melatonin in insect physiology is not fully understood and what constitutes a daily rhythm in insects is not fully defined. In some insects, melatonin and AANAT content fluctuate in a circadian manner, one case being P. americana.68,91 Because of their

47 complex eye structure,94 P. americana are capable of interpreting a photoperiod message as a temporal cue in order to initiate physiological responses that allows them to distinguish between night and day.89 In contrast to P. americana, melatonin and AANAT production failed to follow any circadian patterning in D. melanogaster.13

While the link between circadian rhythms and the photoperiodic system remains largely unknown in insects, previous studies have hinted at a possible link between circadian clock systems and diapause.88,95 Three families of hormones, prothoracicotropic hormone (PTTH), ecdysteroids, and juvenile hormones (JHs) are vital in regulating diapause and pupation. These hormones work in daily rhythms during insect larval stages, and therefore can be categorized as circadian rhythm regulators.96,97 In A. pernyi, melatonin regulates PTTH release, acting as an endocrine switch, thereby, connecting circadian rhythms with endocrine function.98 Mohamed et al. demonstrated that iAANAT is the critical switch regulating PTTH and, subsequently, diapause. By acting as a regulator of this endocrine system, iAANAT may enable a wide variety of insects to maintain homeostasis and circadian function.

It has been seen in few insects that both melatonin synthesis and iAANAT expression follow a circadian rhythm. There are other insects, such as A. pernyi, where iAANAT acts as a mediator of circadian rhythms and endocrine systems through the regulation of PTTH. In this case, of A. pernyi having the ability to interpret photoperiodic responses, melatonin secretion is known to participate in PTTH stimulation. From the evidence presented, we cannot argue definitively that iAANATs are insect “timezymes”.

It is just not clear if iAANATs are directly involved in regulating circadian rhythms like what has been demonstrated in vertebrates. However, iAANATs do seem to mediate rhythmic

48 processes within insects, meaning that iAANATs, and, perhaps; AANATs in general, are more appropriately referred to as ‘rhymezymes’.

2.6 Conclusion

Outlined in this chapter is what is currently known about N-acylated derivatives of the aromatic amino acids and the N-acylated derivatives of the biogenic amines produced in vivo from the aromatic amino acids: dopamine, serotonin, tyramine, tryptamine, phenethylamine, and octopamine. Most, if not all, of these metabolites found in insects are produced enzymatically in a reaction catalyzed by an insect arylalkylamine N- acyltransferase. iAANATs from a number of different insects have been purified and characterized. Detailed studies have established that the kinetic mechanism for many iAANATs is steady-state ordered with the acyl-CoA substrate binding first and catalysis taking place only after the formation of the iAANAT•acyl-CoA•amine complex. An active- site Glu residue found in a highly conserved DEPLN motif is often the general-base critical to iAANAT catalysis. In addition, alanine-scanning mutagenesis of D. melanogaster iAANATs points toward a rate-determining conformational change that may regulate product release. An expanding base of structural information for the iAANATs will only deepen our understanding of iAANAT-mediated catalysis.

The research summarized herein points out some future research directions: the identification of other N-acylated metabolites in insects, defining the function, receptors, and transporters for these molecules in insects, identifying the enzyme responsible for the biosynthesis and degradation of the N-acylated biogenic amines, and the use of mechanistic, sequential and structural information about the iAANATs for the development of iAANAT-specific insecticides to control insect pests. The low sequence –

49 high structural homology has always been a characteristic of insect AANATs. Also apparent is how key conserved residues, such as Arg-153 in DmAANATA and Arg-138 in DmAANATL2, can have different functions in catalysis or structure. This example illustrates that much remains to be learned about the intricate interplay between structure and catalysis for iAANATs. Conformational dynamics are important to substrate binding and catalysis for the GNAT family of enzymes,54,99–101 including the iAANATs.14,102

Solution NMR is an excellent method to study protein dynamics, yet has found little application towards GNAT enzymes.103,104 The iAANATs provide examples for NMR investigations of protein dynamics because these proteins are often monomeric, small

(molecular weights <35 kDa), do not aggregate at mM concentrations, and are expressed at high levels in E. coli. Thus, solution NMR investigations of the iAANATs could yield important new insights into the role of dynamics in the structure/function relationships for the GNAT enzymes.

The role served by the iAANATs in vivo is a source of debate. Due, in part, to the link between mammalian AANATs and the circadian rhythms, it is easy to assume iAANATs play a similar role in insects. It is apparent this is not the case, with each insect, and their corresponding group of iAANATs present a unique model for metabolomics investigations.

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CHAPTER THREE:

IDENTIFICATION OF CATALYTICALLY DISTINCT AANAT SPLICOFORMS FROM

TRIBOLIUM CASTANEUM

3.1 Introduction

The Gcn5-related N-acetyltransferase (GNAT) superfamily of enzymes are known to catalyze the acyl-CoA-dependent acylation of an amine or arylalkylamine;1,2 examples include the arylalkylamine N-acyltransferases (AANATs),3,4 N-acetylglutamate synthetase,5 and the histone acetyltransferases.6 Genomic screens have shown that multiple GNAT-like enzymes could be expressed in an organism.7–9 One possible reason for the heterogeneity of GNAT enzymes in an organism lies in the array of functions mediated by these enzymes.6,10–16 In mammals for example, only one AANAT has been identified, serotonin N-acetyltransferase (SNAT),11 an important regulator of mammalian circadian rhythm. On the other hand, insects express an abundance of AANAT and

AANAT-like enzymes. This is consistent with the understanding that insect AANATs perform a variety of roles, including cuticle sclerotization, biogenic amine removal, and fatty-acid amide biosynthesis.14–18 Much effort has been put into identifying and characterizing these enzymes and the panel of substrates each incorporates, from both an acyl-CoA donor and amine acceptor perspective.7,17,19–33

It has been shown that iAANATs generally show <40% sequence homology but demonstrate remarkable structural conservation;34 four conserved sequence motifs and 68 a common fold core are synonymous with all GNAT enzymes, despite the low pairwise sequence identity.1 Additionally, many iAANATs characterized to date have demonstrated significant variation in activity, especially regarding specificity for the amine acceptor.

While this can be expected between enzymes that share little similarity, it is important to note that even variants of the same gene, expressed through alternative splicing, can serve different metabolic purposes. This has previously been demonstrated for

DmAANATA and DmAANATB, wherein a truncation of 35 amino acids from the N- terminus results in two distinct proteins with differing tissue distribution and instar expression levels.28 Minimal variation in substrate specificity across a panel of biogenic amines tested, however, provided no clear insight into the distinction of these splicoforms.19 As was noted, it is possible that the differences could be due to some unrevealed substrate specificity, or indeed separate modes of regulation between the two enzymes.

Recently, an AANAT from another insect, Tribolium castaneum, was shown to be a key component in cuticle sclerotization and pigmentation.24 The enzyme, TcAANAT1, catalyzes the formation of acetylated amines with dopamine, tryptamine, norepinephrine, tyramine and octopamine functioning as the acceptor substrate. A BLAST search of the

T. castaneum genome using TcAANAT1 as a template identified two separate AANATs, the aforementioned TcAANAT1 and TcAANAT0, the characterization of which is outlined in Chapter 4. Also identified were several isoforms of TcAANAT1. One such isoform is a truncated version of the enzyme, 24 amino acids shorter: TcAANAT1b. We set about examining the effect of truncation on catalytic efficiency and substrate specificity. To do this, we utilized a convenient and straightforward strategy to identify substrates for GNAT

69 enzymes and any other enzymes that utilize acyl-CoA thioesters as acyl donors. This screening strategy allowed us to show that truncation of TcAANAT1 does indeed result in a catalytically distinct version of the enzyme with significantly higher levels of activity.

Through sequence and structural analysis, we then examined the reasoning behind this contrast.

3.2 Results and discussion

3.2.1 Amine screening

A screening strategy, outlined in section 3.4.2, was utilized to determine substrate specificity. A truncated selection of three amine pools tested with acetyl-CoA, all with very different activities, is presented in Table 3.1. Pool 1 and 2 demonstrated activity above the control threshold as defined by Equation 3.1 (Discussed in section 3.4.1). Each amine was then individually examined to determine which acted as substrate for the enzyme.

Table 3.1: Screening results for three distinct pools of amines. All units are in A/min

Pool Pool contents x̅ s σs x̅ c σc Z-Factor Pass or fail?

Lysine Threonine 1 5.4 0.27 0.014 0.001 0.85 Pass Glycine Histamine Serotonin Dopamine 2 0.57 0.08 0.014 0.001 0.56 Pass Tryptamine Norepinephrine Aspartate Isoleucine 3 0.043 0.004 0.014 0.001 0.42 Fail Tryptophan Glutamine

3.2.2 Amine steady-state kinetic constants

In total, eight amines were found to have activity with TcAANAT1b with acetyl-CoA acting as donor: dopamine, octopamine, serotonin, 2-phenethylamine, norepinephrine,

2,2-difluoro-2-phenethylamine, histamine, tryptamine and tyramine. This panel points to

TcAANAT1b having a function in biogenic amine neurotransmitter inactivation. Their respective kinetic constants are presented in Table 3.2 While tryptamine and tyramine were substrates, a strong tendency to inhibit at low concentrations made it difficult to accurately measure rates.

Table 3.2: Steady-state kinetic constants of most active amine substrates for TcAANAT1b. Kinetic constants were measured using 300 mM Tris-HCl, pH 8.0, 375 µM DTDP, and 250 µM acetyl-CoA

-1 -1 -1 Substrate KM (μM) kcat (s ) kcat/KM (M s ) Dopamine 9.7 ± 2.0 26 ± 1.2 (2.7 ± 0.5) x 106 Octopamine 18 ± 5.9 40 ± 5.1 (2.2 ± 0.7) x 106 Serotonin 42 ± 10 70 ± 11 (1.7 ± 0.1) x 106 2-Phenethylamine 81 ± 22 87 ± 14 (1.1 ± 1.1) x 106 Norepinephrine 53 ± 9.9 41 ± 2.5 (7.7 ± 1.5) x 105 2,2-Difluoro-2-phenethylamine 327 ± 43 85 ± 3.8 (2.6 ± 0.4) x 105 Histamine 1,400 ± 190 59 ± 2.8 (4.3 ± 0.6) x 104

Despite other substrates showing a faster turnover rate, the low KM for dopamine means it is likely the endogenous ligand for TcAANAT1b. This is in good agreement with the findings for TcAANAT1, due to its association with cuticle morphology and pigmentation. While both enzymes share five common substrates; dopamine, octopamine, tryptamine, tyramine and norepinephrine, the respective kinetic constants are very different for these enzymes, as shown in Table 3.3.

Table 3.3: Kinetic constants recorded for different variants of TcAANAT1

-1 -1 -1 TcAANAT1 KM (μM) kcat (s ) kcat/KM (M s ) Dopamine 110 ± 40 0.5 ± 0.1 (4.5 ± 0.2) x 103 Octopamine 70 ± 10 0.4 ± 0.06 (5.7 ± 1.2) x 103 Norepinephrine 50 ± 30 0.1 ± 0.03 (2.0 ± 1.3) x 103 -1 -1 -1 TcAANAT1b KM (μM) kcat (s ) kcat/KM (M s ) Dopamine 9.7 ± 2.0 26 ± 1.2 (2.7 ± 0.5) x 106 Octopamine 18 ± 5.9 40 ± 5.1 (2.2 ± 0.7) x 106 Norepinephrine 53 ± 9.9 41 ± 2.5 (7.7 ± 1.5) x 105

While the measurements performed by Noh et al. were made following a different assay, they both used the formation of CoA-SH to track reaction progress. While there are noticeable differences between KM values, this is roughly maintained below a 10-fold difference between the two variants. However, the rate of catalysis is severely affected.

Recorded kcat values are decreased 100 to 400-fold in the longer TcAANAT1 variant. It is apparent that extension of the N-terminus of the enzyme is of serious detriment to the catalytic efficiency of the enzyme. This is through either reducing the binding capacity of acetyl-CoA, or slowing down the rate of product formation, rather than by preventing the amine substrate from binding.

In addition to the catalyic differences between substrates active with both enzymes, a discrete set of amines from the screening assay were found to be active for

TcAANAT1b. Serotonin, 2-phenethylamine, 2,2-difluoro-2-phenethylamine and histamine were not demonstrated to show activity for TcAANAT1 but were substrates for

TcAANAT1b. While a full list of amines tested is not available for TcAANAT1, which therefore makes it difficult to rule out activity for all listed above, the difference in activity demonstrates the two enzymes each have very different catalytic capabilities.

3.2.3 Acyl-CoA steady-state kinetic constants

To examine the capacity of TcAANAT1b to acylate, a full panel of acyl-CoAs were tested for activity while holding the concentration of histamine constant. Histamine was selected because many of the other substrates (namely the catecholamines) were found to have an inhibitory effect at relatively low concentrations. The full panel of acyl-CoAs tested included acetyl-CoA, butyryl-CoA, decanoyl-CoA, palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA, linoleoyl-CoA, arachidonyl-CoA, malonyl-CoA and benzoyl-CoA.

Table 3.4: Steady-state kinetic constants of acyl-CoA substrates for TcAANAT1b. Kinetic constants were measured using 300 mM Tris-HCl, pH 8.0, 375 µM DTDP, and 5 mM histamine

-1 -1 -1 Substrate KM (μM) kcat (s ) kcat/KM (M s ) Acetyl-CoA 65 ± 9.8 52 ± 2.5 (8.0 ± 0.1) x 105 Butyryl-CoA 45 ± 4.9 14 ± 0.5 (3.1 ± 0.4) x 105 Decanoyl-CoA 6.7 ± 0.8 3.4 ± 0.1 (5.1 ± 0.6) x 105

As shown in Table 3.4, activity was found only from acetyl-, butyryl-, and decanoyl-

CoA, demonstrating that TcAANAT1b is exclusively a short-chain acylator, with acetyl-

CoA functioning as the endogenous acyl-donor. Comparison between the two TcAANAT1 variants is not possible with regards acyl-CoA kinetic constants as no such recordings were made for TcAANAT1. In general, iAANATs show much less promiscuity with regards the acyl-CoA substrate. Activity among iAANATs characterized has routinely been shown from acetyl- to decanoyl-CoA with only a small selection demonstrating long-chain acylation. While a decreasing KM compensates for a significantly lowered kcat, it appears the amine binding pocket becomes blocked if the acyl chain is beyond a certain length.

For this reason, it could be assumed that TcAANAT1 shows a similar selection of acyl-

CoA substrates as its truncated variant.

3.2.4 Importance of the AANAT N-terminus

The reasons for the significant increase in catalytic efficiency upon truncation of

TcAANAT1 are as of yet unknown. As shown in Figure 3.1 (underlined), the catalytic site of the amine binding pocket is often located relatively close to the start of the N-terminus in AANATs.

While no crystal structure has been obtained for TcAANAT1, several other iAANAT crystal structures do exist; from T. castaneum (TcAANAT0 – Discussed in Chapter 4), D. melanogaster (DmAANATA),38 and A. aegypti (AaNAT2, AaNAT5b and AaNAT7),39 as well as mammalian SNAT.40,41 SNAT has the longest N-terminus before amine binding pocket, approximately 60 residues long. The conserved structure of this N-terminus is a

β-sheet which then forms an α-helix and makes up part of the amine binding pocket. This binding motif begins at residue 70 in TcAANAT1. It is possible that some conformational change is important at this particular motif to either facilitate acetyl-CoA binding or product formation and release in this enzyme.

Figure 3.1: Sequence alignment of AANAT1 (NCBI reference: XP_972873.2), AANAT1b (NP_001139379.1) and AANAT0 (XP_972873.2) from Tribolium castaneum, as well as AANATA (NP_523839.2) from Drosophila melanogaster

Despite the existence of several insect AANAT structures with ligands bound, no apo-structure has yet been obtained. So, to examine the conformational changes associated with acetyl-CoA binding, the apo and holo structures for SNAT were compared. It is interesting to note that upon acetyl-CoA binding to SNAT, much (~95%) of the structure retains its basic shape except for the N-terminus which undergoes a significant change. A motif stretching from 51 to 67 is a disordered region in the unbound form of SNAT which stretches into the yet unformed amine binding pocket. Upon acetyl-

CoA binding, this organizes into the amine pocket α-helix. This is apparent upon structural alignment of free SNAT (PDB: 1B6B)41 with that bound to the bisubstrate analog CoA-S- acetyl-5-bromotryptamine (PDB: 1KUV)40 (Figure 3.2). The structural similarities between

AANATs implies that a similar mechanism possibly takes place in iAANAT. The extension of the N-terminus in TcAANAT1 may have a detrimental effect on this conformational shift. To examine this further, a crystal structure of the bound and unbound forms of

TcAANAT1 must be obtained.

Figure 3.2: Structure of SNAT with (pink/red) and without (green/blue) bisubstrate analog bound. The blue and red motifs represent residues 51-67 which undergo significant organization upon initial substrate binding.

3.3 Conclusion

Through the use of a well-validated and cost-effective screening assay, we have demonstrated the ability to quickly identify and characterize differences in kinetic ability, not just between different N-acetyltransferases, but between seemingly identical variants of the same N-acetyltransferases. This process allows us to quickly answer the basic questions of catalytic efficiency and begin the process of understanding the intrinsic differences in structure and chemical mechanism which are the root origin for these differences. Using these methods, we have shown that the truncated variant of

TcAANAT1 is a much more catalytically active form of the enzyme. As is often the case for alternative splicing, this is potentially a method to control the rate of acetylation of biogenic amines. In this sense, excess substrate or a scarcity of product results in an upregulation of the truncated form of the enzyme. These findings give further credence to the heterogeneity of insect AANATs.

3.4 Materials and methods

3.4.1 Cloning of TcAANAT1b

The codon-optimized TcAANAT1b gene (accession no. NP_001139379.1) was synthesized by Genscript and ligated using New England Biolabs Quick Ligation™ kit into a pET-28a vector using XhoI and NdeI restriction enzymes. The vector was transformed into E. coli BL21(DE3) competent cells for subsequent protein expression. The transformed cells were cultured at 37°C in 20g/L LB media, supplemented with kanamycin

(50 μg/mL) until an OD600 of 0.6 was achieved. Protein expression was induced with the addition of 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture was left at 30°C for 5 hours. The cultures were then centrifuged at 5000 x g for 10 min at 4 °C and

78 the resulting pellet was resuspended in 20 mM Tris, pH 8.0, 500 mM NaCl, and 5 mM imidazole. The cells were lysed via sonication and centrifuged at 10,000 x g for 20 min at

4°C. The supernatants containing the N-terminal His6-tagged TcAANAT1b was collected and left rocking in 6 mL of HisPur nickel-nitrilotriacetic acid (Ni-NTA) chelating resin. The resin with the bound protein was then added to a column and charged with 10 column volumes (CVs) of 20 mM Tris, pH 8.0, 500 mM NaCl, and 5 mM imidazole followed by a wash of 20 CVs of 20 mM Tris, pH 8.0, 500 mM NaCl, and 60 mM imidazole. The purified

TcAANAT1b was eluted in 1.0 mL fractions with 20 mM Tris, pH 8.0, 500 mM NaCl, and

500 mM imidazole. The protein concentration in each fraction was measured using the

Bradford assay and the protein dialyzed for 16 hours at 4°C against 20 mM Tris pH 7.9,

200 mM NaCl. Following dialysis, the samples were assayed again for concentration and the purity was assessed via SDS-PAGE gel. Purity was determined to be ≥95% continuously for both proteins.

3.4.2 Screening assay

Once the protein was isolated, we began investigating an array of amine substrates available as well as to determine if the enzyme can perform short (acetyl – decanoyl), and long-chain (lauryl – arachidonoyl) acylation. To do this, we catalogued each primary amine available to us, totaling about 90 primary amines. To test these one by one would be time and resource consuming. Instead, each was categorized and pooled in groups of four based primarily on their molecular weight and chemical classification. For example, aminoglycosides were contained in one pool, while catecholamines were similarly pooled together. The final concentration of each amine in the pooled solution was kept constant at 100 mM.

The N-acyltransferase driven acylation of an amine results in two products, the N- acylamide and coenzyme A (CoA-SH). CoA-SH formation can be quantified spectrophotometrically using a reporter molecule with the ability to detect free thiols in biological solutions. 5,5’-dithiobis-(2-nitrobenzoic acid), the well-known Ellman’s reagent,35 has classically been used in this manner. However, more recent studies have used 4,4’-dithiodipyridine (DTDP), noting its greater sensitivity.36 The in vitro formation of

CoA-SH results in reduction of the disulfide bond of DTDP to the corresponding thioketone (Ɛ = 19,800 M-1cm-1 at pH 8.0),36 the rate of which can be measured at 324 nm (Figure 3.3).

Figure 3.3: Reaction of CoA-SH with 4,4’-dithiodipyridine producing a conjugated thioketone detectable at 324 nm (Ɛ = 19,800 M-1cm-1 at pH 8.0) To perform the screening assay, each pool was tested individually at 30°C, with a saturating amount of either acetyl-CoA (short chain: 2 carbons) or oleoyl CoA (long chain:

18 carbons) present. A reaction volume of 750 μL was made up with 300 mM Tris (pH

8.0), 375 μM DTDP, 250 μM acetyl/oleoyl CoA, and 20 mM of the screening pool. The

80 reaction was initiated with 320 nM of enzyme and the rate of change of absorbance per second was recorded.

While a clear hit is apparent with this assay, a statistical parameter for the evaluation and validation of the results is necessary, especially when approaching the threshold for what may or may not be subjectively considered a hit. A simple method for this is to measure the background rate of hydrolysis of the acyl-CoA and set a standard of 10x that rate for what quantifies a hit. To validate this assay further, we measured the ratio of the separation band to the signal dynamic range of the assay. Also known as the

Z-factor,37 this measurement is rudimentarily used to determine the viability of an assay for high-throughput screening. However, we found that it was equally viable in determining a hit from a pool of substrates. A score between 0.5 and 1 indicates a pool that merits further examination. Any value below 0.5 can be considered to not be a hit.

This is shown in Equation 3.1, where σS and σC denote the sample and background signal standard deviation across several replicates, and x̄ S and x̄ C denote the sample and background signal means across several replicates. A control measurement for this assay is important because unavoidable hydrolysis of the acyl-CoA substrate in solution leads to a slight, but consistent rate of CoA-SH formation.

Equation 3.1 (3휎 + 3휎 ) 푍 = 1 − 푠 퐶 |𝐱̄ 푠 − 𝐱̄ 퐶|

A ‘hit’, which constituted a successful screen, saw the individual reagents tested one by one using the same conditions previously mentioned to find the amine(s) responsible for the positive result. This test was repeated for all the screens until a panel of substrates was gathered. From here, individual kinetic parameters were measured 81 using the same spectrophotometric technique, and varying substrate concentration. This method was also used in a similar fashion to test varying acyl chain lengths by holding the concentration of histamine constant, then testing a range of acyl-CoAs to see if any long-chain acyl-CoAs, branched-CoAs, carboxylic-CoAs, or benzylated-CoAs act as donor substrates.

3.5 References

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CHAPTER FOUR:

CHARACTERIZATION OF AANAT FROM TRIBOLIUM CASTANEUM:

A POTENTIAL NEXT-GENERATION INSECTICIDE TARGET

4.1 Introduction

The agricultural industry relies extensively on pesticides in an effort to prevent destruction of field crops and stored crops. The increased demand for crop protection through pesticide application is expected to drive the global agrochemicals market to

$308.92 billion by 2025.1 The issue of pesticide resistance, however, has meant that as a species develops ways to negate the effects of a pesticide, an increased dose must be applied.2 While it is argued that high doses are necessary for complete control over a crop,3,4 this has led to these chemicals entering our food supply at dangerous levels, many of which are known or probable carcinogens,3,8 harming not just mammals,7,8 but also important pollinating species such as the honey bee.9–11 With regards to insect control, many novel insecticides have entered the market with lower toxicity and higher selectivity;12,13 however, their low polypharmacology classically means they are not as economically viable as commercial insecticides, potentially leading to an increased rate of resistance development.14

To combat insecticide resistance, a combination of (1) mixing, rotating, and mandatory breaks in use of currently marketed insecticides, (2) continued design of novel insecticides, and (3) identification of novel enzymatic targets for insecticides must be 89 utilized. A thorough understanding of the mechanism of action of a potential new target will enable the development of inhibitors that will target just one, or a select group of enzymes within a specific insect pest. This will reduce the negative secondary effects on mammals and/or pollinators.

Arylalkylamine N-acyltransferase (often denoted arylalkylamine N- acetyltransferase – AANAT) has shown potential as a novel insecticide target.15,16 These enzymes catalyze the transfer of an acyl group from an acyl-CoA donor to a primary amine acceptor: R1-NH2 + R2-CO-S-CoA → R2-CO-NH-R1 + CoA-SH. This reaction is synonymous with gene transcription regulation via histone acetylation (histone acetyltransferase – HAT),17,18 mammalian circadian rhythm maintenance via serotonin acetylation (serotonin N-acetyltransferase – SNAT), 19,20 and dopamine acetylation, a key step in insect cuticle sclerotization and melanism (dopamine N-acetyltransferase –

DAT).21–25 Additionally, insects lack monoamine oxidase, a regulatory enzyme which facilitates the inactivation and excretion of monoamine neurotransmitters such as dopamine and norepinephrine.26 AANAT is one of a group of enzymes proposed to perform this role in insects.27,28 Recent evidence also suggests that AANATs have a role in the biosynthesis of long-chain fatty acid amides in insects.29,30

As opposed to mammal and avian genomes, where only one AANAT has been identified (SNAT),19,31 insects contain several iAANATs and iAANAT-like (iAANATL) enzymes, perhaps, resulting from the wide range of functions ascribed to the iAANATs in vivo. For example, Drosophila melanogaster and Aedes aegypti have at least eight and thirteen iAANATs/iAANATLs, respectively.32,33 Notably, sequence identity between the iAANATs/iAANATLs is most often <40%.34 The low sequence homology combined with

90 the variety of cellular functions of the iAANATs/iAANATLs suggests that these enzymes are excellent targets for novel insecticide design.17,18

Thorough insight on the mechanistic and structural characteristics of an iAANAT will foster the development of a specific insecticide targeted against that enzyme. This information will allow us to predict structural features that can be built into a putative inhibitor, and to predict how an enzyme may naturally evolve resistance to the inhibitor.

Herein, we present an analysis of one iAANAT from Tribolium castaneum, one of the most common secondary pests that infest and destroy stored grain products worldwide. To date, insecticides such as phosphine, cyanogens, chloropyrifos, and malathion have shown limited success at negating the damage caused by T. castaneum.35–38 These pesticides are not viable long-term solutions for T. castaneum control because of emerging resistance (especially in developing countries) and the detrimental effects these have on the environment.39

Previous work on iAANATs has significantly expanded our understanding of the structure and mechanisms (kinetic and chemical) of these enzymes; however, we are only now beginning to appreciate their variability. Our data show that T. castaneum iAANAT continues this trend of heterogeneity with respect to kinetic and chemical mechanism, substrate specificity, and structure/function characteristics. These appreciable differences highlight the individuality of each AANAT, not just from insect to insect, but also from isoform to isoform within each insect.

4.2 Results and discussion

4.2.1 Acyl-CoA steady-state kinetic constants

A panel of twelve acyl-CoAs were selected to test for activity with TcAANAT0: acetyl-, propionyl-, butyryl-, DL-β-hydroxybutyryl-, crotonyl-, and decanoyl-CoA all represented short-chain acyl-CoAs. DL-β-hydroxybutyryl CoA and crotonyl CoA were used to examine the effect of β-hydroxylation and desaturation on activity respectively.

Palmitoyl, stearoyl- and oleoyl-CoA all represented long-chain acyl-CoAs. Finally, malonyl-CoA and succinyl-CoA represented carboxylate-containing CoAs, and benzoyl-

CoA represented an aryl CoA. Short-chain acyl-CoAs were substrates for TcAANAT0

(Table 4.1), while the long-chain acyl-CoAs and malonyl-CoA were not. Weak activity was also seen for succinyl-CoA, and benzoyl-CoA. Butyryl-CoA was shown to have an ~160- fold decrease in activity when β-hydroxylated but only a 3-fold decrease when unsaturated. Hydroxylation had the greatest effect on acyl-CoA binding, with the main reason for the loss in activity being a sharp increase in KM. Interestingly, a trend of steadily decreasing KM values from acetyl- to decanoyl-CoA was observed, which hints at an active site capable of accepting and binding to longer chain CoAs.

We have observed a similar trend for iAANATs from Drosophila melanogaster and

40,41 Bombyx mori. However, the decreasing kcat value as the length of the acyl chain increases implies that catalysis is not feasible for long-chain acyl-CoA substrates. This likely results from the amine binding pocket becoming sterically occupied upon long-chain acyl-CoA binding, preventing either (a) the amine from binding, (b) the occurrence of a catalytically important conformational change, or (c) improper positioning of the substrates within the active site. Further investigation is needed to uncover the

92 evolutionary and structural divergencies that differentiate iAANATs that accept short- chain acyl-CoAs as substrates from those that accept long-chain acyl-CoA as substrates such as DmAANATL2 and Bm-iAANAT.29,30 This phenomenon is discussed further in

Chapter 6.

Table 4.1: Steady-state kinetic constants of active acyl-CoA substrates for TcAANAT0. Kinetic constants were measured using 300 mM Tris-HCl, pH 8.0, 375 µM DTDP, and 5 mM tryptamine.

-1 -1 -1 Substrate KM (μM) kcat (s ) kcat/KM (M s ) Acetyl-CoA 25 ± 1.8 29 ± 0.6 (1.2 ± 0.1) × 106 Propionyl-CoA 21 ± 2.1 17 ± 0.5 (8.0 ± 0.8) × 105 Butyryl-CoA 15 ± 1.8 5.5 ± 0.1 (3.7 ± 0.5) × 105 Crotonyl-CoA 29 ± 3.5 3.3 ± 0.2 (1.1 ± 0.2) × 105 DL-β-hydroxybutyryl-CoA (1.0 ± 0.4) × 103 2.3 ± 0.5 (2.3 ± 0.1) × 103 Decanoyl-CoA 0.9 ± 0.2 0.8 ± 0.1 (9.6 ± 2.2) × 105 Succinyl-CoA (6.8 ± 1.4) × 102 1.1 ± 0.1 (1.6 ± 0.3) × 103 Benzoyl-CoA 48 ± 6.5 0.04 ± 0.01 (9.3 ± 1.3) × 102

4.2.2 Amino steady-state kinetic constants

A panel of >40 amines were screened against acetyl- and oleoyl-CoA to establish the amine substrate specificity and to determine if the length of the acyl-chain of the acyl-

CoA substrates influences the specificity for the amine. A modified version of the strategy described by Dempsey et al.41 and expanded upon in Chapter 2 was employed to evaluate the amines as TcAANAT0 substrates. The amines used in the screening strategy included amino acids, an array of biogenic catecholamines and indolamines, polyamines, and aminoglycosides. We also included amines with a pKa <8.0, i.e. amines that will possess a deprotonated amine group at the pH used for our screening assay (pH

8.0). Those amines demonstrating activity above the baseline activity level resulting from the non-enzymatic hydrolysis of the acyl-CoA substrate were analyzed to determine their

93 kinetic constants. No activity was observed for any of the amines with oleoyl-CoA as the acyl donor; however, activity was found for 10 amines paired with acetyl-CoA, summarized in Table 4.2. The promiscuity of TcAANAT0 compared to the putative T. castaneum DAT previously studied by Noh et al.42 indicated a more functionalized enzyme, providing a unique insight into AANAT activity.

Table 4.2: Steady-state kinetic constants of most active amine substrates for TcAANAT0. Kinetic constants were measured using 300 mM Tris-HCl, pH 8.0, 375 µM DTDP, and 250 µM acetyl-CoA

-1 -1 -1 Substrate KM (μM) kcat (s ) kcat/KM (M s ) 2-Phenethylamine 140 ± 18 100 ± 8.1 (7.1 ± 1.1) × 105 Dopamine 140 ± 11 61 ± 1.3 (4.4 ± 0.3) × 105 2,2’-Difluoro-2-phenethylamine 340 ± 46 78 ± 4.5 (2.3 ± 0.3) x 105 DL-Norepinephrine 480 ± 46 53 ± 1.8 (1.1 ± 0.1) x 105 Serotonin 480 ± 77 35 ± 2.0 (7.4 ± 0.1) x 104 Histamine 1,700 ± 170 57 ± 1.7 (3.4 ± 0.4) x 104 Tryptamine 260 ± 29 33 ± 0.8 (9.2 ± 0.8) x 104 Tyramine 520 ± 61 36 ± 1.3 (7.0 ± 0.8) x 104 Agmatine 1,200 ± 110 77 ± 3.0 (6.4 ± 0.6) x 104 Octopamine 2,500 ± 140 43 ± 0.8 (1.7 ± 0.1) x 104

4.2.3 Product characterization

The assay we employed to measure activity was dependent upon the release of free CoA-SH.43 Thus, the data of Table 4.1 and 4.2 do not exclude the possibility of an amine-activated thioesterase activity for TcAANAT0. Mass spectrometry was used to characterize the product (N-acetyltryptamine) and confirm TcAANAT0 was indeed synthesizing acylated amines. The retention times and high-resolution mass-to-charge ratios of the enzyme product and the commercial standard are shown in Table 4.3 to validate the catalytic product of the enzyme. A no-enzyme control was used to confirm

94 the thermodynamic formation of N-acetyltryptamine was not occurring independent of the enzyme catalyst at the same rate.

Table 4.3: TcAANAT0 product characterization. Assay conditions – 300mM Tris, pH 8.0, 1mM acetyl-CoA, 1mM tryptamine,100 μg TcAANAT0.

Sample Retention Time (min) m/z Intensity TcAANAT0 assay 3.445 203.1187 266,055.84 N-Acetyltryptamine standard 3.487 203.1188 4,096,377.28 No-enzyme control 3.433 203.1182 1,498.61

4.2.4 TcAANAT0 kinetic mechanism

The kinetic mechanism for TcAANAT0 was examined using a combination of initial kinetics analysis, dead-end inhibition, and direct ligand binding by isothermal titration calorimetry. A kinetic fingerprint for an ordered mechanism was obtained by varying the concentration of acetyl-CoA at different fixed concentrations of tryptamine (Figure 4.1); the data was best fit to an ordered bi-bi mechanism. Our results eliminate the possibility of a random mechanism, which has been attributed to several AANATs previously.40,44

Catalysis, in the case of TcAANAT0, involves the initial binding of acetyl-CoA and only takes place after the formation of a TcAANAT0•acetyl-CoA•tryptamine ternary complex.

Figure 4.1: Acetyl-CoA and tryptamine initial velocity double reciprocal plots. (A) Velocities measured at fixed concentrations of tryptamine: 720 μM (), 360 μM (), 125 μM (), 75 μM () and 50 μM (). (B) Velocities measured at fixed concentrations of acetyl-CoA: 100 μM (), 37 μM (), 20 μM (), and 10 μM (). These data are best fit to the rate equation for a sequential mechanism

Isothermal titration calorimetry was used to confirm the binding of acetyl-CoA to

the enzyme (Figure 4.2). We found that acetyl-CoA binds to free TcAANAT0 with a Kd

vaule of 0.33 ± 0.04 μM. This is in good agreement with the Kd obtained for acetyl-CoA

from Bm-iAANAT3 of 0.66 ± 0.03 μM.45 The considerable enthalpic contribution (ΔH = -

9.49 ± 0.13 kcal/mol) and unfavorable entropic contribution (-TΔS = 0.639 kcal/mol)

indicate that binding of acetyl-CoA significantly stabilizes the structure of TcAANAT0. This

is in agreement with the ITC measurements for the binding of acetyl-CoA to

DmAANATA.46

Figure 4.2: ITC measurements of acetyl-CoA binding to TcAANAT0. Titrations included an initial 0.2 μl injection and 12 subsequent injections of 500 μM acetyl-CoA into 40 μM TcAANAT0, with a 3 min time interval between injections.

Next, to determine the order of substrate binding and product release, a set of

dead-end inhibition experiments was performed. The substrate analogs desulfo-CoA and

tryptophol were used as dead-end inhibitors. Inhibition constants were measured for all

compounds. The data are summarized in Table 4.4 where KI,s is the inhibition constant 97 affecting the slope (when I binds to E), and KI,i is the inhibition constant affecting the intercept (when I binds to ES). The inhibition patterns for desulfo-CoA, competitive vs. acetyl-CoA and non-competitive vs. tryptamine (Figure 4.3), and for tryptophol, competitive vs. tryptamine and uncompetitive vs. acetyl-CoA (Figure 4.4), confirm an ordered sequential mechanism for substrate binding, with acetyl-CoA binding first.

Product inhibition was attempted with N-acetyltryptamine, but no inhibition was detected at a concentration as high as 10 mM. N-Acetylagmatine was also evaluated as a product inhibitor. However, with a measured IC50 >50 mM, the acetylated amine products simply do not bind to the free form of the enzyme, suggesting a conformational change upon acetyl-CoA binding. This conformational change has been shown previously in Bombyx mori iAANAT to facilitate interaction with the incoming amine.45 The lack of inhibition from our product analogs means determination of product release order was difficult. However, ordered product release with the N-acetylamine being released first and free CoA-SH being released second has been observed for other iAANATs and is consistent with both the lack of any appreciable binding of the N-acetylamines to free enzyme, and the ability of desulfo-CoA to do so.41

Table 4.4: Inhibition patterns observed for inhibitors of the amine binding pocket (tryptophol) and acyl-CoA binding pocket (desulfo-CoA). Kinetic constants were measured with varied tryptamine concentrations of 50, 75, 100, 150, 300, and 500 µM and fixed 50 µM acetyl-CoA. Acetyl-CoA concentrations were varied at 10, 20, 30, 50, 80, and 100 µM with fixed 300 µM tryptamine. Tryptophol concentrations were varied at 0, 2 and 4 mM. Desulfo-CoA concentrations were varied at 0, 0.5, and 2 mM.

Inhibitor Varied Substrate Inhibitor Pattern KI,s (mM) KI,i (mM) Tryptamine NC 1.1 ± 0.2 0.6 ± 0.07 Desulfo-CoA Acetyl-CoA C 0.3 ± 0.03 -- Tryptamine C 2.2 ± 0.1 -- Tryptophol Acetyl-CoA UC -- 4.0 ± 0.2

Figure 4.3: Inhibition patterns observed for desulfo-CoA for the amine binding pocket (left) and acyl-CoA binding pocket (right).

Figure 4.4: Inhibition patterns observed for tryptophol of the amine binding pocket (left) and acyl-CoA binding pocket (right).

Finally, we interrogated the TcAANAT0-catalyzed reaction using the bisubstrate analog, CoA-S-acetyltryptamine as an inhibitor. Inhibition plots resulted in competitive and non-competitive apparent KI values of 25 ± 1.9 μM and 120 ± 3.7 μM against acetyl-

CoA and tryptamine respectively (Figure 4.5). However, the observance of multiple rates of product formation, rather than one initial steady-state rate followed by a plateau indicated slow-binding inhibition. We measured the rate of product formation for four concentrations of inhibitor (20, 30, 100 and 200 μM), across a time frame where a steady- state rate is observed in the absence of inhibitor (30 sec) (Figure 4.6). The true KI value

true 47 (KI ) was found to be 16 ± 1.7 μM following the methods of Schlesinger et al. and

Merkler et al.48 (see Section 4.4.4). The mechanism of inhibition is shown in Figure 4.7 and the data summarized in Table 4.5.

Figure 4.5: Inhibition patterns observed for CoA-S-acetyltryptamine. Kinetic constants were measured with varied tryptamine concentrations of 50, 75, 100, 150, 300, and 500 µM and fixed 50 µM acetyl-CoA. Acetyl-CoA concentrations were varied at 10, 20, 30, 50, 80, and 100 µM with fixed 300 µM tryptamine. CoA-S-acetyltryptamine concentrations were varied at 0, 100 and 250 µM.

100

The time-dependence in the inhibition of this bisubstrate analog indicates a

structural reorganization upon formation of the intermediate. This is akin to a kinetic

proofreading mechanism, wherein the active site affinity for the substrates increases upon

intermediate formation, slowing the reaction down and assuring correct product

formation.49

Figure 4.6: Time-dependent inhibition of TcAANAT0 by CoA-S-acetyltryptamine. (A) Plot of progress curves of TcAANAT0 saturated with acetyl-CoA and tryptamine and with varying concentrations of inhibitor. (B) Activity regeneration of TcAANAT0 after 10- minute incubation with inhibitor compared against no-incubation control. (C) Plot of kobs vs. [I].

101

Table 4.5: Summary of kinetic rate and inhibition constants attributed to time-dependent inhibition

Kinetic Parameter app -1 -1 1 k5 (M s ) (7.2 ± 1.1) × 10 true -1 -1 2 k5 (M s ) (7.9 ± 1.2) × 10 -1 -2 k6 (s ) (1.2 ± 0.02) × 10 Half-life (τ) (s) 83 ± 1.2 KI,app, acetyl CoA (μM) 25.3 ± 1.9 true KI (μM) 16.2 ± 1.7

Figure 4.7: Kinetic model for slow-binding inhibition. Initial inhibitor binding (k3) is true followed by a second slower step (k5) induced by a structural reorganization. KI is * defined by the ratio of k5 and k6, the relative formation and relaxation of EI , the maximally inhibited complex.

102

4.2.5 Crystal structure and mutagenesis of TcAANAT0

In order to get a better structural understanding of TcAANAT0, we attempted to crystalize the apo protein. However, crystallization efforts with the apo TcAANAT0 proved unsuccessful after several attempts. We next attempted to co-crystallize TcAANAT0 with acetyl-CoA, in the hopes that the acetyl-CoA could stabilize the protein enough to allow for crystal formation. The crystal structure of TcAANAT0 in complex with acetyl-CoA was determined at 2.84 Å resolution and belongs to the C2 space group. The crystal has six copies of TcAANAT0 in the asymmetric unit, with each copy bound to acetyl-CoA. The

Fo-Fc electron density for acetyl-CoA is well-defined with the exception of the pantetheine tail, where the density is weaker than the rest of the compound. The adenine portion of acetyl-CoA forms a prominent stacking interaction with Phe-166 and another acetyl-CoA present in an adjacent TcAANAT0 molecule (Figure 4.8). Notably, no other AANAT structure has shown such an interaction, highlighting the heterologous nature of iAANATs. Creation and analysis of the F166A mutant illustrated the functional importance of this residue, as the F166A mutant is a catalytically inert form of the enzyme (Table 4.6)

These stacking interactions between adjacent acetyl-CoA molecules, and Phe-166, likely served to stabilize the lattice and helped to induce crystal formation. This possibility is further evidenced by the lack of crystal formation when TcAANAT0 was not co-crystallized with acetyl-CoA.

103

Figure 4.8: Crystal Structure of TcAANAT0 in complex with acetyl-CoA. The complex x- ray crystal structure of TcAANAT0 in complex with acetyl-CoA is shown at 2.84 Å resolution. The unbiased Fo-Fc electron density map is contoured at 3σ and shown in green. TcAANAT0 is colored in magenta, and acetyl-CoA is colored in orange. Hydrogen bonds are shown as black dashed lines.

Further examination of the binding of acetyl-CoA to TcAANAT0 demonstrates favorable interactions with multiple active site residues (Figure 4.9). Specifically, the three phosphates in acetyl-CoA form several potential hydrogen bonds with the TcAANAT0 backbone in the area from Gly-135-Lys-140, as well as with the Lys-140 side chain. The pantothenic acid portion of acetyl-CoA also establishes extensive interactions with the active site. Notably, Arg-134 forms a hydrogen bond with acetyl-CoA, in addition to van der Waals interactions between the acetyl-CoA geminal methyl groups and the Arg-134 104 side chain. Arg-134 aligns with Arg-153 of DmAANATA41, Arg-138 of DmAANAT250, and

Arg-138 of DmAANAT751, which have been shown to be important in maintaining the structure of the acyl-CoA binding pocket. Production of the R134A mutant of TcAANAT0 confirmed this, with a 94% and 88% drop in catalytic efficiency for acetyl-CoA and tryptamine. A 15-fold increase in KM and preservation of kcat for acetyl-CoA is testament to the structural rather than catalytic importance of Arg-134 (Table 4.6).

Figure 4.9: Structure of acetyl-CoA binding interactions within the TcAANAT0 active site. Acetyl-CoA is shown in pink and interacting residues shown in gray.

105

Sequence alignment with other characterized iAANATs also indicated that Glu-25 located in close proximity to the amine binding pocket and the acetyl group of the bound acetyl-

CoA holds catalytic relevance in TcAANAT0 (Figure 4.10). This is due to its correlation to

Glu-47 of DmAANATA,41 Glu-26 of DmAANAT7,51 Glu-34 of DmAgmNAT52 and Glu-27 in Bm-iAANAT3,40 which have all been shown to act as a general base during catalysis.

Figure 4.10: Primary sequence alignment showing conserved Glu residue (within amine binding pocket motif DEPLN) attributed to functioning as a general base in insect AANATs.

Kinetic analysis of an E25A mutant of TcAANAT0 was in similar agreement, illustrated by a 92% and 98% drop in catalytic efficiency for acetyl-CoA and tryptamine respectively. Surprisingly, production of an E25D conserved mutant yielded similar results, with a 78% and 95% drop in catalytic efficiency recorded for acetyl-CoA and tryptamine respectively (Table 4.6). It is interesting to note that shortening the length of the residue R-group by one methylene group significantly deters the ability of the enzyme to perform catalysis. 106

Table 4.6: Steady-state kinetic constants for TcAANAT0 site-directed mutants. Kinetic constants were measured in triplicate as previously described for the wild-type enzyme

Acetyl-CoA Mutant -1 -1 -1 KM (μM) kcat (s ) kcat/KM (M s ) % Effectiveness Wild-Type 25.0 ± 1.8 29 ± 0.6 (1.2 ± 0.1) x 106 100 E25A 12.0 ± 2.4 1.1 ± 0.1 (9.7 ± 2.1) x 104 3.8 E25D 8.7 ± 1.8 2.3 ± 0.1 (2.6 ± 0.5) x 105 22.1 R134A 394 ± 22 29 ± 0.7 (7.4 ± 0.4) x 104 6.3 F166A N/A N/A N/A 0 Tryptamine Mutant -1 -1 -1 KM (μM) kcat (s ) kcat/KM (M s ) % Effectiveness Wild-Type 260 ± 29 33 ± 0.8 (9.2 ± 0.8) x 104 100 E25A 751 ± 46 1.40 ± 0.1 (1.8 ± 0.1) x 103 4.1 E25D 500 ± 68 2.2 ± 0.1 (4.5 ± 0.6) x 103 4.8 R134A 1130 ± 75 12.8 ± 0.2 (1.1 ± 0.1) x 104 12.2 F166A N/A N/A N/A 0

4.2.6 TcAANAT0 chemical mechanism

To determine the chemical mechanism of TcAANAT0, the pH-dependence of binding and catalysis was measured for tryptamine (Figure 4.11), 2,2-difluoro-2- phenethylamine (DFPE) (Figure 4.12), and acetyl-CoA (Figure 4.13). Rate profiles allowed the determination of likely catalytic or structurally important residues based on their apparent pKa values. The fits produced profiles with three ionizable groups involved in binding and/or catalysis (Table 4.7). An acidic pKa of approximately 7.3 was attributed to a general base, which has classically been found in iAANATs.41,50,53 The exact function of this general base has been argued, but it has been proposed that deprotonation of the amine occurs prior to formation of a tetrahedral intermediate.41 To investigate this, rate profiles were generated for 2,2’-difluoro-2-phenethylamine (DFPE; predicted pKa ≈ 6.7 ±

0.3 – calculated via SciFinder using Advanced Chemistry Development (ACD/Labs)

Software 11.02). The disappearance of the acidic limb when measuring kcat against pH

107 highlights that at pH levels of 6 to 8, where DFPE is still largely found in its deprotonated form (when compared to tryptamine, pKa ≈10.2),54 the function of the general base is negated.

Figure 4.11: pH rate profiles for tryptamine illustrating the effect of pH on kcat, and kcat/KM.

Figure 4.12: pH rate profiles for DFPE illustrating the effect of pH on kcat, and kcat/KM.

Figure 4.13: pH rate profiles for acetyl-CoA illustrating the effect of pH on kcat, and kcat/KM.

108

A second pKa of approximately 9.0 was also observed, with evidence indicating two unresolvable species are involved. The first of these can likely be attributed to the

54 thiol group of the departing CoA-SH, which has a reported pKa ≈ 10.2. This pKa has also been attributed to a general acid, which facilitates the release of the products. However, after careful examination of the TcAANAT0 crystal structure, there is no obvious active site residue that might function as a general acid during catalysis. We propose rather than a general acid actively involved in catalysis, a more likely scenario involves a residue, upon amine binding, forming a hydrogen bond with the general base and acting as a polarizer. This would stabilize the charge on the glutamate and increase its pKa, giving credence to a glutamate performing a general base role.

Table 4.7: Summary of pKa values measured from pH rate profiles.

Acidic Limb pKa values Basic Limb pKa values

-1 -1 kcat (s ) kcat (s ) Acetyl CoA 7.4 ± 0.1 Acetyl CoA 9.6 ± 0.3 Tryptamine 7.1 ± 0.2 Tryptamine 8.3 ± 0.2 DFPE N/A DFPE 8.7 ± 0.1

-1 -1 -1 -1 kcat / KM (M s ) kcat / KM (M s ) Acetyl CoA 6.5 ± 0.2 Acetyl CoA 8.7 ± 0.3 Tryptamine 7.9 ± 0.1 Tryptamine 9.3 ± 0.2 DFPE 7.5 ± 0.2 DFPE 8.6 ± 0.2

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We propose two plausible chemical mechanisms for TcAANAT0. In the first mechanism (Figure 4.1), Glu-25 is the base responsible for the deprotonation of the amine moiety of tryptamine, nucleophilic attack at the carbonyl of the thioester of acetyl-

CoA generates a zwitterionic tetrahedral intermediate, and an unidentified general acid acts to polarize Glu-25. Subsequent protonation of CoA-SH results in breakdown of this intermediate in a time-dependent manner, as illustrated by the time-dependent inhibition of TcAANAT0 by CoA-S-acetyltryptamine. This time-dependence also points to intermediate collapse likely being the rate-determining step. Upon product release, this

‘acidic’ residue moves out of hydrogen bond range of the base, thereby relaxing the pKa of the base resulting in its deprotonation and reformation of the enzyme active site. The

Figure 4.14: Proposed chemical mechanism for T. castaneum AANAT0 with an unidentified general acid responsible for the identified pKa of 9.8.

110 role of a general acid has been suggested for AANATs previously (as discussed in

Chapter 2), however, thus far, researchers have been unable to directly correlate one residue to this role. It is possible that the acid has a passive, rather than active role in catalysis. If the general acid is part of a motion after amine binding, it would stand that the corresponding residue may not be obvious from crystal structures.

Figure 4.15: Proposed chemical mechanism for T. castaneum AANAT0 with the departing CoA-SH responsible for the identified pKa of 9.8.

The second proposed mechanism (Figure 4.15) follows a very similar vein to that proposed for other iAANATs.41,46,50,52 This mechanism differs only from that shown in

Figure 4.14 in that the pKa of 9.8 is solely attributed to the departing CoA-SH rather than

111 a general acid. Due to the data indicating two unresolvable pKa values ≈9.8, we favour the former mechanism for TcAANAT0.

4.3 Conclusion

TcAANAT0, a previously uncharacterized AANAT from Tribolium castaneum, was found to have significant promiscuity in both acyl-CoA donor and amine acceptor, as opposed to the previously characterized TcAANAT1. The kinetic mechanism follows a steady-state ordered mechanism, with acyl-CoA binding first. Generation of a crystal structure of TcAANAT0 bound to acetyl-CoA allowed us to pinpoint some key features in the enzyme active site, which, coupled with site-directed mutagenesis and pH-rate profiling, allowed us to shine further light on the chemical mechanism of iAANATs. The knowledge gained from such intrinsic understanding of the mechanistic and structural relationship of iAANATs is invaluable in their development as insecticide targets.

Fragment-based drug discovery in the pursuit of AANAT-targeted inhibitors with insect specificity are in progress.

4.4 Experimental procedures

4.4.1 Materials

Unless otherwise noted, all reagents were of the highest quality available from commercial sources. Codon-optimized TcAANAT0 was purchased from Genscript.

Oligonucleotides were purchased from Eurofins MWG Operon. BL21 (DE3) E. coli cells,

XL-10 competent cells, and the pET-28a(+) vector were purchased from Novagen.

PfuUltra High-Fidelity DNA polymerase and the QuikChange Site-Directed Mutagenesis

Kit were purchased from Agilent. XhoI, NdeI, Antarctic phosphatase, and T4 DNA ligase were purchased from New England Biolabs. Kanamycin monosulfate and isopropyl-β-D-

112 thiogalactopyranoside (IPTG) were purchased from Gold Biotechnology. ProBond nickel- chelating resin was purchased from Invitrogen. Acyl-CoAs, N-acetyltryptamine, and the amine substrates were purchased from Sigma-Aldrich. Desulfo-CoA was synthesized in lab following the methods of Chase, et al.54 CoA-S-acetyltryptamine was synthesized in lab following the methods of Khalil and Cole.55 Spectrophotometric analyses were performed on a Cary 300 Bio UV-Visible spectrophotometer. Isothermal titration calorimetry was performed on a VP-ITC calorimeter (GE Healthcare).

4.4.2 Cloning of T. castaneum AANAT0

The codon-optimized genes for T. castaneum AANAT0 (NCBI reference sequence: XM_967780.4) was cloned, overexpressed and purified following the procedure described in Chapter 3, Section 3.4.1.

Additional purification steps were necessary for crystallization. Following affinity purification, the protein was dialyzed against a buffer of 50 mM Tris HCl, pH 8.0, and 10 mM CaCl2 in preparation for thrombin cleavage of the His6-tag. Protein (10 mg) was incubated with thrombin agarose resin at 4°C for 24 hours following the recommended procedure. The sample was then loaded onto a Ni-NTA column and the cleaved protein eluted in 1 mL fractions with 20 mM Tris, pH 8.0, 500 mM NaCl, and 60 mM imidazole.

SDS-PAGE was used to check the purity of the eluted protein. The appropriate fractions were combined and concentrated using a Millipore ultrafiltration cell. The concentrated sample was further purified via FPLC using a SuperDex 75 gel filtration column. The running buffer was 20 mM Tris pH 7.4, 50 mM NaCl, 1 mM EDTA, and 1 mM DTT and the final purified protein was concentrated to 16.9 mg/mL.

113

4.4.3 Determination of enzyme activity

4,4’-Dithiodipyridine (DTDP) was used to determine the activity of TcAANAT0 at

324 nm (molar absorptivity = 19,800 M-1cm-1)56 by measuring the formation of CoA-SH product. Each 750 μL assay consisted of 300 mM Tris, pH 8.0, 375 μM DTDP, and the desired concentration of acyl-CoA and amine substrates. Amine and acyl-CoA kinetic constants were measured by holding the concentration of acetyl-CoA and tryptamine at fixed, saturated levels (250 μM and 5 mM) respectively. Measurements were made in triplicate using a Cary 300 Bio UV−visible spectrophotometer at 30 °C. Steady-state kinetic parameters were evaluated by fitting initial velocity measurements to Equation 4.2 using Enzyme Kinetic Wizard (SigmaPlot 12.0).

Equation 4.2

푉푚푎푥[푆] 푉표 = 퐾푀 + [푆]

4.4.4 Determination of kinetic mechanism

To fully define the kinetic mechanism of TcAANAT0, a combination of initial velocity double-reciprocal analysis and dead-end inhibition was utilized. Firstly, initial velocities were measured by varying the concentration of one substrate while holding the other substrate at constant, but different concentrations. Measurements were made at acetyl-CoA concentrations of 10, 20, 37, and 100 μM, and tryptamine concentrations of

50, 75, 125, 360, and 720 μM. The resulting data were fit to Equation 4.3 for an ordered bi-bi mechanism (χ2 = 27.5) using Visual Enzymics 2010 (IGOR Pro 6.34 A).

Equation 4.3 푉 [퐴][퐵] 푚푎푥 퐾푖,푎퐾푀,푏 + 퐾푀,푎[퐵] + 퐾푀,푏[퐴] + [퐴][퐵]

114

To further define the kinetic mechanism, tryptophol and desulfo-CoA were utilized as dead-end inhibitors for tryptamine and desulfo-CoA respectively. Initial velocity patterns were obtained by varying the concentration of one substrate, while holding the concentration of the second at approximately the KM. This was then repeated twice in the presence of fixed concentrations of inhibitor. To test inhibition of the amine binding site, tryptophol was used as the inhibitor at 2.0 and 4.0 mM. To test inhibition of the acyl-CoA binding site, desulfo-CoA was introduced as the inhibitor at 500 and 1000 μM.

Measurements were made at varying tryptamine and acetyl CoA concentrations of 50,

75, 100, 150, 300 and 500 μM, and 10, 20, 30, 50, 80 and 100 μM respectively. To measure the steady-state inhibition of CoA-S-acetyltryptamine, measurements were made at varying tryptamine and acetyl CoA concentrations of 50, 75, 100, 150, 300 and

500 μM, and 20, 30, 50, 80, 100 and 120 μM respectively and at 0, 100 and 250 μM CoA-

S-acetyltryptamine. The data were then fit to Equation 4.4, Equation 4.5, and Equation

4.6 using Enzyme Kinetic Wizard (SigmaPlot 12.0) to differentiate between competitive, non-competitive, and uncompetitive inhibition.

Equation 4.4 푉 [푆] 푚푎푥 퐼 퐾푀 (1 + ) + [푆] 퐾푖푠

Equation 4.5 푉 [푆] 푚푎푥 퐼 1 퐾푀 (1 + ) + [푆] (1 + ) 퐾푖푠 퐾푖푖

115

Equation 4.6 푉 [푆] 푚푎푥 1 퐾푀 + [푆] (1 + ) 퐾푖푖

To measure the time-dependence of CoA-S-acetyltryptamine, the rate of reaction was measured at 3 mM tryptamine and 250 μM acetyl-CoA. Enzyme concentration was kept low to ensure the longest possible steady-state duration in the absence of inhibitor.

Consecutive measurements were then made at 20, 30, 100 and 200 μM CoA-S- acetyltryptamine. The resulting progress curves were fit using SigmaPlot 12.0 to Equation

4.7, where vi is the initial rate, and vf is the final rate in order to calculate kobs, the observed first-order rate constant. The rate constant of dissociation, koff, was measured directly via jump dilution. Enzyme at 40 μM was incubated for 10 min. with 250 μM of CoA-S- acetyltryptamine. After incubation, 1 μL of this mixture was then used to initiate the reaction, resulting in a 750-fold dilution of inhibitor. These data were then fitted to

Equation 4.7, with vi measured as 1.0 μmoles/min and vf measured as 7.1 μmoles/min.

app This allowed kon to be estimated using Equation 4.8 under the assumption of competitive inhibition against acetyl-CoA binding. A good fit indicated a one-step slow

app binding mechanism. The resulting estimate, kon was adjusted to account for substrate

true true competition using Equation 4.9 to give kon . Finally, the true inhibition constant, KI was calculated using Equation 4.10.

116

Equation 4.7

(푣푖 − 푣푓) −푘표푏푠푡 [푃] = 푣푓푡 + ( ) (1 − 푒 ) 푘표푏푠

Equation 4.8

푎푝푝 푘표푏푠 = 푘표푛 [퐼] + 푘표푓푓

Equation 4.9

푡푟푢푒 푎푝푝 [푆] 푘표푛 = 푘표푛 (1 + ) 퐾푀

Equation 4.10

푡푟푢푒 푎푝푝 푘표푓푓 퐾퐼 = 퐾퐼 ( 푡푟푢푒) 푘표푛

4.4.5 Determination of acetyl-CoA binding constants

Isothermal titration calorimetry (ITC) was used to investigate the interaction between acetyl-CoA and TcAANAT0. Experiments were performed on a VP-ITC calorimeter (GE Healthcare) at 30°C and using. Following Ni-NTA affinity chromatography purification, as described previously, TcAANAT0 was dialyzed overnight against 20 mM

Tris, pH 8.0, 100 mM NaCl, 0.5 mM EDTA and 1 mM tris(2-carboxyethyl)phosphine

(TCEP). Enzyme samples were then diluted to 40 μM and loaded into a 200 μL sample cell. Acetyl-CoA samples of ~10-fold excess were prepared from the same dialysis buffer and loaded into a 40 μL injection syringe. Titrations included an initial 0.2 μl injection and

12 subsequent injections of acetyl-CoA until completion, with a 3 min time interval

117 between injections. The data were fit using Origin 7.0 (OriginLab Corporation) with the point of initial injection excluded and using an on-site binding model.

4.4.6 Determination of pH-dependence

pH rate profiles were generated to measure the pH-dependence of TcAANAT0 with regards to acetyl CoA binding. Kinetic constants were measured at 0.25 pH increments from pH 5.75 to pH 7.0 and 0.5 pH increments from pH 7.0 to pH 9.5 using the following buffers: MES (pH 5.75 – 6.75), TRIS (pH 7.0 – 8.5) and CHES (pH 9.0 – pH

10.0). The data were fit using Visual Enzymics 2010 (IGOR Pro 6.34 A) Equation 4.11 for a monobasic system with a slope of -2 indicating two unresolvable hydrogen ion dissociation constants, and Equation 4.12 and Equation 4.13 for a dibasic system with an acidic leg slope of -1 and -2 respectively. Here, y represents the kinetic constant measured, either KM, kcat, or kcat/KM, C represents the maximum value for the kinetic constant, H represents the hydrogen ion concentration, and Ka, K1 and K2 represent the hydrogen ion dissociation constants. All fits were subjected to Levenberg-Marquardt optimization to determine optimal fit.

Equation 4.11 퐶 푦 = (퐾 )2 (1 + 푎 ) 퐻2

Equation 4.12 퐶 푦 = 퐻 퐾 (1 + + 2) 퐾1 퐻

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Equation 4.13 퐶 푦 = 퐻 퐾2 퐾1퐾2 1 + + + 2 퐾1 퐻 퐻

4.4.7 Product characterization

Mass spectrometry was used to demonstrate that the kinetic activity recorded was due to the formation of the acylamine product, and not simply due to hydrolysis of the acyl-CoAs. The product was first generated by incubating 100 μg of TcAANAT0 with 3.5 mM tryptamine, 0.3 mM acetyl-CoA, and 300 mM Tris, pH 8.0 at 30°C for 10 minutes.

The enzyme was filtered out using a Millipore 10 kDa spin column and the remaining solution containing the enzymatic product was diluted 500-fold. 20 μL was injected into a

Phenomenex Kinetex 2.6 μm C18 100 Å (50 mm × 2.1 mm) reverse phase column coupled to an Agilent 6540 liquid chromatography/quadrupole time-of-flight mass spectrometry

(LC/QTOF-MS) in positive ion mode as described previously by Dempsey et al.30 A commercial standard of N-acetyltryptamine was also run following the same method. The retention times and high-resolution mass-to-charge ratios of the enzyme product and the commercial standard were then compared to validate the catalytic product of the enzyme.

4.4.8 TcAANAT0 mutagenesis

To investigate the importance of individual residues in substrate binding and catalysis, alanine scanning was performed on several residues which have classically shown to be important in AANAT activity or are shown via crystal structure to be of interest. The mutants made were: E25A, E25D, R134A, and F166A. Primers for these mutants were designed using the Agilent QuikChange Primer Design tool, and the mutants were synthesized using an Agilent QuikChange Lightning kit following the

119 recommended procedure. Following transformation, all mutant DNA was sequenced to confirm successful mutation. The mutant enzymes were cultured, purified and kinetically analyzed following the same procedures as described above.

4.4.9 TcAANAT0 crystallization

Crystal screening was performed using an Art Robbins Instruments Phoenix Liquid

Handling robot and several commercially available screens from Qiagen. Hits identified from screening were scaled up. TcAANAT0 was crystallized in 200 mM sodium citrate pH

5.5, and 20% PEG 3000, at 20°C using hanging drops in a 1:1:1 ratio

(protein:buffer:acetyl-CoA). Single crystals formed after 3-4 days and grew to full size in

6-7 days. The crystals were harvested and cryo-cooled using a solution of the crystallization buffer with 20% glycerol and 10 mM acetyl-CoA added.

4.4.10 X-ray data collection and refinement

Data were collected using the 19-BM beamline of the Structural Biology Center

(SBC) at the Advanced Photon Source, Argonne, Illinois. Data was processed using iMosflm, and the CCP4 suite was used to refine the data. Coot was used to build and

57-59 60 refine the complete model. PyMol was used to generate all protein structure figures.

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CHAPTER FIVE:

Bm-iAANAT3: EXPRESSION AND CHARACTERIZATION OF A NOVEL

ARYLALKYLAMINE N-ACYLTRANSFERASE FROM BOMBYX MORI

Note to Reader: Portions of this chapter has been previously published in Archives of

Biochemistry and Biophysics, 2019, 661, 107-116 and has been reproduced as permitted by Elsevier (See Appendix A).

5.1 Introduction

N-Acetylamides, CH3-CO-NH-R, have been isolated and characterized from a host of vertebrates and invertebrates and are usually produced in vivo by a reaction between

1–5 acetyl-CoA, CoA-S-CO-CH3, and the corresponding amine, R-NH2. The enzymes catalyzing amine N-acetylation are the arylalkylamine N-acetyltransferases (AANATs, also known as arylalkylamine N-acyltransferases)6,7 which are members of the Gcn5- related N-acetyltransferase enzyme superfamily.8–10 In insects, the iAANATs (iAANATs = insect AANATs) produce N-acetylserotonin, an intermediate in melatonin biosynthesis, and are also thought to function in controlling melanism,11,12 the inactivation of biogenic amines,13,14 the sclerotization of the insect cuticle,14,15 and photoperiodism.7 Our interest in the AANATs is derived from our work on the biosynthesis of the fatty acid amides,16,17 a family of cell signaling lipids.18 We and others have found that AANAT-like (AANATL) enzymes will catalyze the formation of fatty acid amides from fatty acyl-CoA thioesters and the corresponding amines.17,19–21 Database searches of insect genomes indicate that 130 a number of iAANATs and iAANATLs could be expressed in a variety of insects.7,14,22

Thus, insects should serve as model organisms to better understand the possible role played by the iAANATs and iAANATLs in the biosynthesis of N-acetyl- and N- acylarylalkylamides and to further define the biological function of these molecules. Much remains unknown about the cellular function of the long-chain N-acylarylalkylamides and other fatty acid amides.23

Herein, we report on the over-expression in E. coli, purification, and characterization of recombinant Bm-iAANAT3, a third iAANAT from B. mori. B. mori express two other iAANATs, Bm-iAANAT and Bm-iAANAT2.24,25 Our work on Bm- iAANAT3 contributes to an understanding of N-acyltransferase chemistry and will foster the development of inhibitors targeted against insect N-acyltransferases, due to their emergence as intriguing targets for insecticide design.26–28 In addition, we plan to extend our mechanistic work on the AANATs to focus on structural elucidation by NMR and X- ray crystallography. The other iAANATs we have over-expressed have thwarted such experiments because these enzymes precipitate at high protein concentrations.20,21,29–32

Bm-iAANAT3 does not precipitate at high protein concentration enabling structural studies of this enzyme by NMR.33,34

5.2 Results and Discussion

5.2.1 Cloning, expression, and purification of Bm-iAANAT3

Bm-iAANAT3 from Bombyx mori was successfully cloned, expressed, and purified from E. coli using a codon-optimized gene, yielding 10.9 mg of Bm-iAANAT3 purified protein per liter of culture. Purity was ≥95% as assessed by SDS-PAGE and the molecular weight of the protein was in good agreement with its mass, 23.9 kDa for Bm-iAANAT3.

131

Bm-iAANAT3 is a previously uncharacterized enzyme, and shares 32%, 24%, and 30% sequence identity with Bm-iAANAT, Bm-iAANAT2, and Dm-AANATA, respectively

(Figure 5.1). It shares slightly less identity with the mammalian AANATs from human and sheep, at 21% and 17% identity, respectively. These data provide additional evidence that AANATs typically share poor sequence homology when compared against each

Figure 5.1: Homology overlays of iAANAT enzymes from Bombyx mori and Drosophila melanogaster. The sequence data used in creating this figure are: D. melanogaster AANATA (Dm-AANATA, accession number is NP_523839), B. mori iAANAT (Bm- iAANAT, accession number is NP_001073122.1), B. mori iAANAT2 (Bm-iAANAT2, accession number is XP_004928436.2), and B. mori iAANAT3 (Bm-iAANAT3, accession number is NP_001177771.1). other, even between enzymes from organisms of the same phylum and class.9

5.2.2 Identification of amine and acyl-CoA substrates for Bm-iAANAT3

We have developed a screening strategy employing different pools of various amine substrates (arylalkylamines, polyamines, aminoglycosides, ethanolamine, histamine, and amino acids; (Table 5.1) that enables the rapid identification of substrates for any putative N-acyltransferase. We have successfully utilized this strategy to identify substrates for N-acyltransferases from Drosophila melanogaster.29–32

132

Table 5.1: Pooled amine substrates for the initial screening assay

Concentration Pool of Each Amine Amino Donors (mM) 1 60 Lysine, Threonine, Glycine, Histamine, and Dopamine 2 60 Alanine, Arginine, Histidine, Tyramine, and Norepinephrine 3 60 Serine, Proline, Methionine, Serotonin, and Tryptamine 4 20 Glutamate, Asparagine, Valine, Ethanolamine, and Octopamine 5 20 Aspartate, Isoleucine, Tryptophan, and Glutamine 6 60 Leucine, Phenylalanine, γ-Aminobutyric acid, and Taurine 7 20 Spermidine, Agmatine, Cadaverine, Putrescine, and β-Alanine

In applying this strategy to identify amine substrates for Bm-iAANAT3, a relatively high concentration of each amine (20 mM or 60 mM, Table 5.1) was included in the pool because the KM value for glycine is reported at 6 mM for some of the glycine N- acyltransferases.35–37 Pools of acyl-CoA substrates were not constructed because of the reports of long-chain acyl-CoAs acting as AANAT inhibitors;29,37,38 therefore, we individually cross-screened amine pools against archetypal short and long-chain acyl-

CoA thioesters, acetyl-CoA and oleoyl-CoA, because both have been reported as substrates for other GNAT family acyltransferases.20,21 For Bm-iAANAT3, we found no significant velocities with oleoyl-CoA above the low enzyme-independent, background rate of oleoyl-CoA hydrolysis, suggesting that this enzyme does not catalyze the formation of long-chain N-acylamides. Employing acetyl-CoA as substrate, a broad range of amines were substrates exhibiting high rates of catalysis far above our background threshold rate of 0.1 μmoles/min/mg. Six arylalkylamines (tryptamine, tyramine, dopamine, octopamine, serotonin, and norepinephrine) and histamine were identified as

133 substrates from the four amine pools that yielded positive results. Ranking the amine substrates from the highest to the lowest kcat/KM values is as follows: tryptamine, tyramine, dopamine, octopamine, serotonin, norepinephrine, and histamine (Table 5.2).

Table 5.2: Kinetic Constants for the Amine Substrates for Bm-iAANAT3a

Amine KM kcat kcat/KM Relative (μM) (s-1) (M-1 s-1) kcat/KM Tryptamine 97 ± 8.8 60 ± 1.2 (6.2 ± 0.6) × 105 33 Tyramine 63 ± 6.7 27 ± 0.6 (4.3 ± 0.5) × 105 23 Dopamine 330 ± 23 55 ± 1.1 (1.6 ± 0.1) × 105 8.4 Octopamine 18 ± 0.8 2.2 ± 0.02 (1.2 ± 0.05) × 105 6.3 Serotonin 1100 ± 63 59 ± 1.0 (5.2 ± 0.3) × 104 2.7 Norepinephrine 140 ± 8.6 5.3 ± 0.07 (3.7 ± 0.2) × 104 1.9 Histamine 1700 ± 98 33 ± 0.68 (1.9 ± 0.1) × 104 1.0

5 -1 The kcat/KM value for the best amine substrate, tryptamine = (6.2 ± 0.6) × 10 M s-1, is ~30-fold higher than the value for the worst amine substrate, histamine = (1.9 ± 0.1)

4 -1 -1 × 10 M s . Octopamine displayed the lowest KM value of 18 ± 1 µM, while histamine displayed the highest KM of 1.7 ± 0.1 mM. None of the polyamines, aminoglycosides, or amino acids we tested served as substrates for Bm-iAANAT3. Interestingly, acetyl-CoA displayed the highest KM value of the acyl-CoA substrates,90 ± 4 µM. The other four acyl-

CoA substrates, buytryl-CoA, hexanoyl-CoA, octanoyl-CoA, and decanoyl-CoA exhibited nearly identical KM values of 10 to 14 µM (Table 5.3). As we have observed with other

20,29–32 insect AANATs, the (kcat/KM)acyl-CoA values decreased as the length of the acyl chain increased for the acyl-CoA substrates: the (kcat/KM)acetyl-CoA is 170-fold higher than the

(kcat/KM)decanocyl-CoA. Decanoyl-CoA was the acyl-CoA substrate with the longest acyl-chain that showed rates of CoA-SH formation that were significantly above the background rate of enzyme-independent acyl-CoA hydrolysis. In addition, we found that malonyl-CoA is a

134 substrate for Bm-iAANAT3 with a (kcat/KM)malonyl-CoA that is ~6-fold lower than the

(kcat/KM)decanocyl-CoA.

Table 5.3: Kinetic constants for the CoA thioester substrates (R-CO-S-CoA) for Bm- iAANAT3. Measurements were made at one, fixed initial concentration of tryptamine. Reactions were run at 22°C in 300 mM Tris pH 8.0, 150 µM DTNB, 8.0 mM tryptamine, and the acyl-CoA concentration was varied in the range 0.3 to 3.0 × KM

Acyl-CoA R KM kcat kcat/KM Relative (μM) (s-1) (M-1 s-1) kcat/KM Acetyl CH3- 90  3.6 64  0.7 (7.1  0.3) × 105 170 Malonyl HOOC-CH2- 1900  300 13  1.5 (7.0  1.3) × 103 0.17 Butyryl CH3(CH2)2- 14  0.8 4.0  0.04 (2.9 ± 0.2) × 105 70 Hexanoyl CH3(CH2)4- 13 ± 0.5 1.3 ± 0.01 (9.5 ± 0.4) × 104 23 Octanoyl CH3(CH2)6- 10 ± 1.0 0.36 ± 0.005 (3.4  0.3) × 104 8.1 Decanoyl CH3(CH2)8- 12 ± 5.5 0.05 ± 0.004 (4.2  1.9) × 104 1.0

To the best of our knowledge, this is the first report of malonyl-CoA serving as a substrate for a GNAT enzyme. N-Succinylation, however, has been demonstrated in the detoxification of mammalian xenobiotics,39,40 N-succinyl conjugates of octopomine, serotonin, dopamine, and tyramine have been identified in C. elegans,41 N- succinylserotonin (bufobutanoic acid) is found in the venom of various toad species (the traditional Chinese medicine, Ch’an Su).42 Crystallographic results also identified succinyl-CoA bound in two GNAT enzymes in addition to the kinetic activity noted for succinyl-CoA with TcAANAT0, noted in chapter 4.43,44 Our data showing that malonyl-

CoA is a Bm-iAANAT3 substrate in combination with these other works hints that a panel of N-malonylated and N-succinoylated amines may exist in both vertebrates and invertebrates.

Our work on Bm-iAANAT3 follows earlier studies of two other iAANATs from B. mori, Bm-iAANAT and Bm-iAANAT2.24,25 One of these, Bm-iAANAT, exhibited a wide 135 expression pattern and accepted a diverse set of amines as substrates for N- acetylation.24 The combination of its broad tissue distribution and amine substrate promiscuity led to the suggestion that Bm-iAANAT is the major enzyme of monoamine catabolism in B. mori.24 The N-acetylation of dopamine is of particular significance in monoamine catabolism. The production of N-acetyldopamine is a reaction critical to sclerotization of the cuticle in insects.15 Dopamine is required for the formation of the brown pigment, dopamine melanin, while N-acetyldopamine is the precursor for a colorless pigment.45 In fact, B. mori defective in dopamine biosynthesis have a colorless and improperly formed cuticle.46 The N-acetylation of serotonin is one of the reactions in the conversion of tryptophan to melatonin.2,6 Since Bm-iAANAT catalyzes the acetylation of both serotonin and dopamine, the enzyme is likely involved in melatonin and dopamine melanin production in B. mori. Bm-iAANAT mutants exhibit higher than normal levels of dopamine and widespread black pigmentation in the larvae and adult moths.11,46 Later, a second B. mori iAANAT was discovered and characterized, Bm-iAANAT2.25 Transgenic flies suppressing Bm-iAANAT2 expression exhibited elevated dopamine levels and melanin deposition in the head and integument. These results suggest that Bm- iAANATL2 contributes to the regulation of melanism in B. mori.

Our substrate specificity studies of Bm-iAANAT3 show that this enzyme is a

“versatile generalist” capable of acylating a broad range of amines, with a preference for acetyl-CoA as a co-substrate. All amines included in Table 5.2, apart from tryptamine, have been identified in B. mori;47–51 thus, it is possible that Bm-iAANAT3 does catalyze their acetylation in vivo. Information regarding the cellular concentration of the amines we have identified as Bm-iAANAT3 substrates is limited. The hemolymph concentrations of

136 dopamine, tyramine, and serotonin from the fourth instar, fifth instar, and pupa stages are low, <10 μM,47 but it is not clear how these concentrations relate to their respective intracellular concentrations. There are two reports of the brain concentrations of biogenic amines in B. mori, all reported as pg/brain.47,48 If the volume of a B. mori brain is ~1 μL, the highest concentration of dopamine and serotonin would be ~0.8 mM. These estimated intracellular concentrations for dopamine and serotonin are comparable to the KM values we determined for these amines, KM for dopamine = 0.33 mM and KM for serotonin = 1.1 mM (Table 5.2). Thus, Bm-iAANAT3 could have a role in the acetylation of dopamine and serotonin in vivo; therefore, functioning in sclerotization, pigmentation, and melatonin production.

5.2.3 Kinetic mechanism

Two different experimental approaches were used to deduce the kinetic mechanism of Bm-iAANAT3. One set of experiments was to vary one substrate, acetyl-

CoA or tryptamine, while holding the other substrate at a fixed initial concentration. Our initial velocity data were subjected to global non-linear regression analysis, to differentiate between several bi-bi kinetic mechanisms.52 The data fit equally well to Equation 3 for a steady-state ordered kinetic mechanism and Equation 4 for a ping-pong mechanism; Chi- squared values for the fits to equations 3 and 4 were the same within experimental error.

An equilibrium ordered kinetic mechanism would yield one plot of 1/v vs. 1/[substrate] that intersect on the 1/v-axis, clearly inconsistent with the data shown in Figure 5.2.

137

Figure 5.2: Bm-iAANAT3 double-reciprocal analysis of initial velocities for acetyl- CoA and tryptamine, fit to equation 3 in a global non-linear regression analysis. (A) Velocities measured at fixed concentrations of tryptamine: 500 μM (), 250 μM (), 120 μM (◆), 60 μM () and 40 μM (). (B) Velocities measured at fixed concentrations of acetyl-CoA: 200 μM (), 90 μM (), 40 μM (◆), 25 μM () and 15 μM ().

138

A pattern of parallel lines for the plot of 1/v vs. 1/[substrate] at fixed concentrations of the second substrate is often indicative of a ping-pong kinetic mechanism; however, a steady-state ordered kinetic mechanism can yield a parallel pattern if the KM for substrate

A is greater than the dissociation constant for substrate A.52 This is exactly what we found

34 for acetyl-CoA, KM = 90 μM and Kd = 0.7 μM.

Next, a set of dead-end inhibition studies were performed to provide additional support for an ordered kinetic mechanism. Oleoyl-CoA and tyrosol were used as dead- end inhibitors against acetyl-CoA and tryptamine, respectively. Neither analog exhibited a rate of reaction above the baseline rate of hydrolysis, and both have been previously utilized as dead-end inhibitors in studies of the D. melanogaster AANATs and mammalian serotonin N-acetyltransferase.29,31,32,38 Oleoyl-CoA was competitive vs. acetyl-CoA

(Figure 5.3A) and noncompetitive vs. tryptamine (Figure 5.3B) with a Ki,s value of 220 ±

20 nM and Ki,s = Ki,i values of 790 ± 30 nM, respectively. Tyrosol was uncompetitive vs. acetyl-CoA (Figure 5.3C) and competitive vs. tryptamine (Figure 5.3D), with a Ki,i value of

230 ± 10 μM and a Ki,s value of 90 ± 10 μM, respectively. Furthermore, these data indicate that enzymatic generation of N-acetyltryptamine from acetyl-CoA and tyramine as catalyzed by Bm-iAANAT3, most likely, occurs via an ordered sequential mechanism with acetyl-CoA binding first followed by the binding of tryptamine to yield the catalytically competent Bm-iAANAT3•acetyl-CoA•tryptamine ternary complex. An ordered sequential kinetic mechanism has been attributed to several D. melanogaster AANATs and GNAT enzymes.29–32,53–56

139

A B

C D

Figure 5.3: Dead-end inhibition plots for Bm-iAANAT3. (A) Velocities measured at a fixed concentration of tryptamine (100 μM) with varied concentrations of acetyl-CoA (10 μM, 25 μM, 50 μM, and 100 μM), at varied concentrations of the inhibitor, oleoyl-CoA: 0 nM (⚫), 250 nM (), and 500 nM (); Ki = 220 ± 20 nM (B) Velocities measured at a fixed concentration of acetyl-CoA (100 µM) with varied concentrations of tryptamine (50 μM, 100 μM, 150 μM, and 300 μM), at varied concentrations of the inhibitor, oleoyl-CoA: 0 nM (⚫), 250 nM (), and 500 nM (), Ki = 790 ± 30 nM (C) Velocities measured at a fixed concentration of tryptamine (100 µM) with varied concentrations of acetyl-CoA (10 μM, 25 μM, 50 μM, and 100 μM), at varied concentrations of the inhibitor, tyrosol: 0 μM (⚫), 150 μM (), and 300 μM (); Ki = 230 ± 10 μM (D) Velocities measured at a fixed concentration of acetyl-CoA (100 M) with varied concentrations of tryptamine (50 μM, 100 μM, 150 μM, and 300 μM), at varied concentrations of the inhibitor, tyrosol: 0 μM (⚫), 150 μM (), and 300 μM (); Ki = 90 ± 10 μM

140

Product inhibition is one method useful in defining the order of product release. N-

Acetyltryptamine produced competitive and noncompetitive inhibition plots against acetyl-

CoA and tryptamine, respectively (Figure 5.4). These data are most consistent with an ordered sequential product release, with CoA-SH being released first followed by release of the acetylated product, N-acetyltryptamine. This is similar to the order of product release reported for N-myristoyltransferase.56 We found that order of product release for

D. melanogaster agmatine N-acetyltransferase (DmAgmNAT) was the N-acetylated product first followed by CoA-SH,32 similar to that reported for human histone acetyltransferase (HAT) and Enterococcus faecium aminoglycoside acetyltransferase

(AAC((6’)-I).53,54 The product inhibition patterns for Bm-iAANAT3 (Figure 5.4) differed from those reported for DmAgmNAT, HAT, and AAC((6’)-I.

Figure 5.4 Product inhibition of Bm-iAANAT3 by N-acetyltryptamine. (Left) Initial velocities measured at a fixed concentration of tryptamine (100 μM), varying the concentrations of acetyl CoA, and varying the concentration of the inhibitor, N- acetyltryptamine: 0 mM, 1.0 mM, 5.0 mM; KI,i = 640 ± 60 mM. (Right) Initial velocities measured at a fixed concentration of acetyl CoA (100 μM), varying the concentrations of tryptamine, and varying the concentration of the inhibitor, N-acetyltryptamine: 0 mM, 1.0 mM, 5.0 mM. KI,s = 4.2 ± 0.2 mM.

141

A steady-state ordered bi-bi kinetic mechanism with acetyl-CoA binding first and

N-acetyltryptamine being released last (Figure 5.6) is consistent with the binding data of

Aboalroub et al..34 Direct binding measurements by ITC and/or NMR showed that acetyl-

CoA and N-acetyltryptamine bound to Bm-iAANAT3 with Kd values of 0.7 ± 0.03 μM and

12 ± 3 μM. The binding of N-acetyltryptamine was eliminated by the addition of acetyl-

CoA and was not affected by the addition of CoA-SH. The binding of tryptophol, the non- substrate alchohol analog of tryptamine, was significantly enhanced by the presence of acetyl-CoA, the Kd value decreasing from 230 ± 12 μM to 50 ± 2 μM. Lastly, CoA-SH binds to Bm-iAANAT3 with low affinity, a Kd of 30 ± 1 μM. Relatively weak binding of tryptamine, Kd = 220 ± 8 μM, and CoA-SH to free Bm-iAANAT indicates that kinetic mechanism is actually random bi-bi with a strong preference for acetyl-CoA binding first and N-acetyltryptamine being released last.

5.2.4 pH dependence of the kinetic constants and identification of a catalytic base

We determined the pH-dependence of the kinetic constants for acetyl-CoA to aid in our identification of amino acids important in catalysis and substrate binding in Bm- iAANAT3. Both the kcat, and (kcat/KM)acetyl-CoA pH-rate profiles exhibited a pH-dependent rise from pH 6.5 to 8.0 following by a pH-independent plateau above pH 8. These data are best fit to equation 8 with a pKa value of 7.3 ± 0.1 for both profiles (Figure 5.5), pointing towards a catalytically important base with a pKa of 7.3. This pKa value likely corresponds to the general base deprotonating the primary amine of tryptamine; alternatively, it could be attributed to the zwitterionic tetrahedral intermediate that is produced after the nucleophilic attack of the acetyl-CoA thioester by tryptamine. This set of experiments did not uncover a second, higher pKa value; this additional pKa (~8.5 – 10.0) would

142 correspond to a general acid in catalysis, routinely observed for NAT enzymes.9,31,57 We hypothesize that Bm-iAANAT3 either lacks a general acid in catalysis, or the general acid has a pKa value > 9.5, a number too high for us to ascertain due to limitations of acetyl-

CoA hydrolysis and/or DTNB instability at elevated pH ranges.

(

Figure 5.5: pH rate profiles for Bm-iAANAT3. (Top) Log(kcat)versus pH for acetyl-CoA. (Bottom) Log(kcat/KM) versus pH for acetyl-CoA.

143

5.2.5 Identification of a catalytic base and a potential chemical mechanism for

catalysis

Alignment of the amino acid sequence of Bm-iAANAT3 to those for other insect

AANATs shows that Glu-27 of Bm-iAANAT3 corresponds to Glu-47 of AANATA from D. melanogaster (Figure 5.1). Dempsey et al. have shown that Glu-47 is likely the catalytic base in that enzyme.29 Thus, we mutated Glu-27 to Ala in Bm-iAANAT3. The E27A mutant enzyme was cultured, purified, and characterized in the same manner as the wildtype Bm-iAANAT3. The kinetic constants we measured for the E27A mutant were:

-2 -1 KM,acetyl-CoA = 20 ± 2.2 μM, kcat,acetyl-CoA = 4.0 ± 0.09 × 10 s , KM,tryptamine = 9400 ± 990 μM,

-2 -1 and kcat,tryptamine = 8.0 ± 0.3 × 10 s . Thus, the E27A mutant retained only 0.06% to

0.13% of the wild-type kcat while exhibiting 100-fold increase in the KM for tryptamine. The significant decline in catalytic activity as compared to wild-type Bm-iAANAT3, in addition to the large perturbation in the KM,tryptamine, suggests that this residue functions as a general base in catalysis, as well as playing a role in substrate binding. Glu-27 likely represents the pKa value of 7.3 observed in the pH-rate profile (Figure 5.5). The ~1000- fold decrease in kcat value for the E27A mutant falls in the range of values measured for general base mutants of other N-acyltransferases in GCN5 family (Table 5.4). A larger kcat effect was measured after the mutation of the catalytic base in mandelate racemase,

58 a ~10,000-fold decrease in the kcat of the D270N mutant.

Given the results for the D270N mutant in mandelate racemase, the general base mutants of Bm-iAANAT3 and the other N-acyltransferases in Table 5.4 have surprisingly high kcat values. Most likely, this results from another chemical species serving as a base in catalysis. Perhaps, an active site water molecule of the “proton-wire” found in the active

144 site of this family of enzymes or another active site amino acid can serve as the “back- up” during catalysis in the absence of the preferred catalytic base.8 Partial rescues of the general base mutants of Dm-AANATL7,31 DmAgmNAT,32 and yeast-HAT at high pH point towards an active site hydroxide serving as the catalytic base.59 In addition, Scheibner et al. argue that His-120 is the catalytic base in serotonin N-acetyltransferase and that His-

57 122 is catalytically redundant, serving as the base in the H120A mutant. While the kcat is only slightly depressed for the individual H120A and H122A mutants ~3-5-fold, the kcat for the double mutant, H120A/H122A, is significantly lower, decreased ~280-fold (Table

5.4).

Table 5.4: Ratio of (kcat,wildtype)/(kcat,mutant) for general base mutant enzymes. Dm = Drosophila melanogaster and Bm = Bombyx mori. Glu-29 is most likely not the catalytic base for Dm-AANATL2.

a Enzyme Mutation (kcat,wildtype)/(kcat,mutant) Ref. DmAgmNAT E34A ~2100 32 Bm-iAANAT3 E27A ~1000 This work Yeast-HAT E173Q ~320 at pH 7.5 58 ~6 at pH 9.5 Dm-AANATL7 E26A ~22 31 Dm-AANATA E47A ~13-170 29,60 Ovine-SNAT H120A ~5 56 H122A ~3 H120A/H122A ~280 Dm-AANATL2b E29A ~1.0 30

When taken together, our data strongly supports the mechanism depicted in Figure

5.6. The double-reciprocal plots and dead-end inhibition analysis indicate an ordered sequential mechanism: acetyl-CoA binds first, followed by tryptamine, leading to the Bm- iAANAT3•acetyl-CoA•tryptamine ternary complex before catalysis occurs. After the binding of both substrates into the active site, the positively charged amine moiety of

145 tryptamine is deprotonated by Glu-27 (the catalytic base) through an ordered set of water molecules. The amine deprotonation step is followed by nucleophilic attack of the deprotonated amine at the carbonyl of the acetyl-CoA thioester to generate a tetrahedral intermediate. The zwitterionic tetrahedral intermediate collapses as the coenzyme A thiolate is protonated by the positively charged amine of the intermediate to yield the two products: CoA-SH and N-acetyltryptamine. Our product inhibition studies show that product release is ordered with CoA-SH exiting first, followed by N-acetyltryptamine.

Figure 5.6: Proposed mechanism for the Bm-iAANAT3-catalyzed N-acetylation of tryptamine.

146

5.3 Conclusion

We have cloned, over-expressed in E. coli, and characterized a previously unknown iAANAT from B. mori, Bm-iAANAT3. The kinetic mechanism for Bm-iAANAT3 with acetyl-CoA and tryptamine is bi-bi steady-state ordered with acetyl-CoA binding first and N-acetyltryptamine released last. Studies of the chemical mechanism suggest that

Glu-27-mediated deprotonation of the amine substrate facilitates nucleophilic attack of the amine at the carbonyl of the acetyl-CoA thioester. Collapse of the resulting tetrahedral intermediate immediately leads to product release, the N-acetylated amine likely leaving first, followed by the CoA-SH by-product.

Substrate specificity data reveal that Bm-iAANAT3 prefers short-chain acyl-CoA thioesters with acetyl-CoA as the best acyl-CoA substrate. While fatty acid amides have been recently identified in B. mori,21 Bm-iAANAT3 is probably not involved in their biosynthesis in vivo. Bm-iAANAT3 accepts a variety of amines as substrates pointing towards a cellular role for this enzyme in amine inactivation and/or excretion. In addition,

Bm-iAANAT3 may have role in sclerotization, melanism, and melatonin biosynthesis by catalyzing dopamine and serotonin acetylation. Our identification of malonyl-CoA as a

Bm-iAANAT3 substrate suggests that a series of biologically-occurring N-malonylated and N-succinoylated amines await discovery and characterization; a suggestion consistent with the identification of N-succinoylated amines in C. elegans.41 Why B. mori would express three iAANATs, Bm-iAANAT, Bm-iAANAT2, and Bm-iAANAT3, with potentially overlapping cellular functions is unknown. Future Bm-iAANAT3 knockdown- experiments in B. mori are planned to address the question of its cellular function.

147

5.4 Experimental procedures

5.4.1 Materials

Unless otherwise noted, all reagents were of the highest quality available from commercial sources. Purchasing details are noted in Chapter 4, Section 4.3.1.

5.4.2 Cloning of Bm-iAANAT3

Bm-iAANAT3 (Accession no. NM_001190842.1) was cloned, overexpressed and purified following the procedure described in Chapter 3, Section 3.4.1.

5.4.3 Substrate identification for Bm-iAANAT3

An activity-based screening protocol was used to identify substrates for Bm- iAANAT3. First, the enzyme was evaluated using acetyl-CoA (representative of a short- chain CoA) or oleoyl-CoA (representative of a long-chain CoA) separately, with different groups of pooled amines. The groups of amines are listed in Table 5.1. Each assay consisted of 300 mM Tris pH 8.0, 150 μM DTNB (Ellman’s reagent, 5,5’-dithiobis(2-nitrobenzoic acid), 500 μM acyl-CoA, and individual amines at 20 mM or 60 mM. Initial velocities were determined by measuring the release of CoA at 412 nm using Ellman’s

-1 -1 reagent (ε412 = 13,600 M cm ) and were calculated after the background rate of CoA thioester hydrolysis was subtracted from the observed velocity. Any amine pool that displayed a background corrected rate ≥0.1 μmoles/min/mg (≥0.04 M-1 s-1) in combination with acetyl-CoA or oleoyl-CoA was further evaluated to identify the specific Bm-iAANAT3 substrate(s) contained within that pool. Each amine within that group was individually interrogated at a relatively high concentration in the presence of acetyl-CoA or oleoyl-

CoA to determine if the amine was truly a substrate for Bm-iAANAT3. The assay conditions were identical to that described for the screening protocol using the amine

148 pools. Individual amines that displayed a background corrected rate of ≥0.1

μmoles/min/mg were included in our measurements for additional kinetic analysis.

5.4.4 Determination of the steady-state kinetic constants

Steady-state kinetic characterization of Bm-iAANAT3 was carried out using

Ellman’s reagent to measure the release of CoA under the following conditions: 300 mM

Tris pH 8.0, 150 μM DTNB, and varying concentrations of acyl-CoA and amine substrates at 22°C.

To determine the kinetic constants for the acyl-CoA substrates, the initial tryptamine concentration was 8.0 mM, while varying the concentration of the acyl-CoA substrates. To determine the kinetic constants for the amine substrates, the initial acetyl-

CoA concentration was 500 μM, while varying the amine.

The kinetic constants for the acyl-CoA and amine substrates for Bm-iAANAT3 were determined by fitting the resulting data to Equation 5.1 using SigmaPlot 12.0: vo represents initial velocity, Vmax is the maximal velocity, KM is the Michaelis Menten constant, and [S] is the concentration of the varied substrate. Note that the concentration of the other substrate was fixed at relatively high (saturating) concentration. Assays were performed in triplicate and the uncertainty for the kcat and kcat/KM values were calculated using Equation 5.2, with σ as the standard error.

Equation 5.1

Vmax[S] vo = KM + [S]

149

Equation 5.2

2 2 x x σ σy σ ( ) = √( x) + ( ) y y x y

5.4.5 Kinetic mechanism for Bm-iAANAT3

5.4.5.1 Double reciprocal analysis

To differentiate between sequential and classic ping-pong kinetic mechanisms, double-reciprocal plots of the initial velocity data were generated for acetyl-CoA and tryptamine in SigmaPlot 12.0. Non-linear regression analysis of initial rates and model discrimination analyses were performed in WaveMetrics IGOR Pro 6.34A. Initial velocities were determined by varying the concentration of one substrate, while holding the other substrate at a fixed concentration. The two plots were generated by holding the tryptamine concentration constant (40 μM, 60 μM, 120 μM, 250 μM, and 500 μM) and varying the concentration of acetyl-CoA. Accordingly, the second plot held the concentration of acetyl-CoA constant (15 μM, 25 μM, 40 μM, 90 μM, and 200 μM) and varied the tryptamine concentration. The resulting initial velocity data was fit to Equation

5,3 for an ordered bi-bi mechanism and to Equation 5.4 for ping-pong mechanism, where vo is the initial velocity, Vmax is the maximal velocity, [A] is the concentration of substrate

A, [B] is the concentration of substrate B, KIa is the dissociation constant for substrate A,

Kb is the Michaelis Menten constant for substrate B, and Ka is the Michaelis Menten constant for substrate A. Kia, the dissociation constant for acetyl-CoA, was fixed at 0.7

μM, based on the direct measurement of acetyl-CoA binding to Bm-iAANAT3 by isothermal calorimetry (ITC).34

150

Equation 5.3

Vmax[A][B] vo = KIaKb + Ka[B] + Kb[A] + [A][B]

Equation 5.4

V [A][B] max Ka[B] + Kb[A] + [A][B]

5.4.5.2 Dead-end inhibition with oleoyl-CoA, tyrosol, and N-acetyltryptamine

To further define the kinetic mechanism of Bm-iAANAT3, we performed dead-end and product inhibition analysis using oleoyl-CoA, tyrosol, and N-acetyltryptamine. The initial velocities were generated by holding one substrate (either acetyl-CoA or tryptamine) at a fixed concentration, varying the concentration of the other substrate, at different fixed concentrations of each inhibitor. All assays were performed in triplicate, and the resulting data was fit to Equation 5.5, Equation 5.6 and Equation 5.7 in SigmaPlot

12.0 for competitive, noncompetitive, and uncompetitive inhibition. Here, vo is the initial velocity, Vmax is the maximal velocity, KM is the Michaelis Menten constant, [S] is the substrate concentration, [I] is the inhibitor concentration, and KI,s is the dissociation constant for the dissociation of I from the E•I complex, and KI,i is the dissociation constant for the dissociation of I from the E•S•I complex.

151

Equation 5.5 V [S] v = max o [I] KM (1 + ) + [S] KI,s

Equation 5.6 V [S] v = max o [I] [I] KM (1 + ) + [S] (1 + ) KI,s KI,i

Equation 5.7 V [S] v = max o [I] KM + [S] (1 + ) KI,i

5.4.5.3 pH dependence of the steady-state kinetic constants

The pH dependence of the kinetic constants was determined for both tryptamine and acetyl-CoA at intervals of 0.5 pH units ranging from pH 6.0 to 9.5. The buffers used were the following: MES (pH 6.0 – 6.5), Tris (pH 7.0 – 9.0) and AmeP (pH 9.0 – 9.5). The resulting kinetic data were fit to Equation 5.8 and Equation 5.9 in WaveMetrics IGOR Pro

6.34A to delineate the pKa values of measured ionizable groups. In both equations, c is the pH-independent plateau.

Equation 5.8

pKa−pH log(kcat/KM) = log[c/(1 + 10 )]

Equation 5.9

pKa−pH log kcat = log[c/(1 + 10 )]

152

5.4.6 Cloning, expression, and purification of site-directed mutants

To further investigate the role of individual amino acids potentially involved in substrate binding and catalysis, a pair of Bm-iAANAT3 mutants was produced: E27A and

S167A. The generation of both mutants was carried out using the overlap extension method.61 Mutants were amplified with the PfuUltra High-Fidelity DNA polymerase with the following PCR conditions: an initial denaturation step of 95°C for 2 minutes, followed by 30 cycles of PCR amplification (95°C for 30 s, 58°C for 30 s, 72°C for 1 min), and a final extension step of 72°C for 10 minutes. Primers were designed on the Agilent

QuickChange Primer Design tool and purchased from Eurofins MWG Operon. PCR products were digested with NdeI and XhoI restriction enzymes and ligated into the pET28a vector with DNA ligase acquired from New England Biolabs. Mutant enzymes were expressed and purified in the same manner as wild type Bm-iAANAT3.

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163

CHAPTER SIX:

WHAT REMAINS TO BE UNEARTHED?

ACTIVE-SITE DISORDER HOLDS KEY TO LONG-CHAIN ACYLATION BY AANAT

6.1 Introduction

The transfer of an acyl group from an acyl-CoA donor to a hydroxyl, thiol, phosphate or amine of some biologically relevant acceptor is of vital importance in nature and is found in essentially every living organism. While it is correct to call all enzymes that perform this function ‘acyltransferases’, historically, research has tended to focus on acetylation. This is due to the widespread prominence of acetylation in metabolism, such as in glycolysis,1 protein post-translational modification,2,3 gene transcription,4–6 and circadian rhythm.7,8 Due to the significance of acetylation,9 the enzymes which govern its catalysis are separated from other forms of acylation into their own class: the acetyltransferases.

Indeed, the concept of acylation is primarily reserved for protein fatty acylation:10 the transfer of a fatty acyl group (upwards of eight carbons) such as myristate and palmitate to an acceptor,11,12 usually to the N-terminal and N-epsilon positions of peptides and proteins. Protein fatty acyltransferases have been shown to enhance membrane binding as well as mediate protein-protein interactions.13–15 However, the discovery of N- arachidonylethanolamine (anandamide),16 the endogenous ligand for the cannabinoid receptor in 1992 introduced the field of fatty acid amide research, focused on elucidating 164 the role of fatty acid amides in cell signaling. The panel of known fatty acid amides quickly grew to include not just the N-acyl amino acids synonymous with protein fatty acylation, but also the N-acylethanolamines,17,18 N-acylarylalkylamides,19,20 and the primary fatty acid amides.21 Indeed, several studies have demonstrated the presence of panels of these long-chain compounds in mammals,22 Drosophila melanogaster,23 and Bombyx mori.24

The biosynthetic pathways of many of these species remains largely unknown,25 however there are two primary pathways that have been suggested. These include a condensation reaction between the corresponding fatty acid and amine substituents catalysed by fatty acid amide hydrolase (FAAH),26 and the transfer of the fatty acyl group from a CoA donor to an amine acceptor catalyzed by the corresponding N- acyltransferase.27 The former of these is unlikely, as it was found that several long-chain

N-acylserotonins are, in fact, inhibitors for FAAH.28 It was therefore hypothesized that a family of N-acyltransferases exist which catalyze the biosynthetic pathway of these fatty acid amides.

6.2 AANATs to fill the gap?

The Gcn5-related N-acetyltransferase (GNAT) superfamily holds some possible candidates for fatty acid amide synthesis.29 This group of enzymes is perhaps most well- known for the acetylation of specific lysine resides on histones,30 catalyzed by histone acetyltransferase, an important regulator of gene transcription. In the context of fatty acid amide synthesis however, attention has turned to one group of GNAT enzymes, known as the arylalkylamine N-acetyltransferases (AANATs). In mammals, AANAT catalyzes the

N-acetylation of serotonin to form N-acetylserotonin.31,32 Subsequent methylation by

165 acetylserotonin-O-methyltransferase forms melatonin, an important regulator of the sleep-wake cycle.7,33,34

Just one AANAT has been discovered in mammals so far: serotonin N- acetyltransferase. However, it remains possible that another AANAT-like enzyme exists, which catalyzes the formation of endogenous fatty acid amides. To determine the possibility of this, more readily available model organisms were implemented; primarily insects. While mammals contain a solitary identified AANAT, insects harbor numerous discrete AANATs. For context, Drosophila melanogaster, the fruit fly, expresses eight putative AANATs, while Aedes aegypti, the yellow-fever mosquito, expresses at least thirteen.20,35 It seemed feasible that were an AANAT to exist in insects which is involved in the biosynthesis of fatty acid amides, a similar enzyme likely exists in mammals.

The role of AANATs in insects is a much more indiscriminate one. They are associated with the formation of N-acetyldopamine, an important aspect of cuticle sclerotization in many insects.36 The distinct lack of monoamine oxidase (MAO) in insects also imparts the function of bioactive amine inactivation upon AANATs.37–39 Numerous labs, including our own, have contributed tirelessly to expressing and characterizing

AANATs from numerous different insects, both structurally and functionally. This list includes the aforementioned D. melanogaster20,23,27,40–45 and A. aegypti,35,46,47 and additionally Bombyx mori,24,48–52 Periplaneta americana,53–55 Acyrthosiphon pisum56 and

Tribolium castaneum.57 It was discovered that these AANATs universally utilized acetyl-

CoA as a donor for traditionally a large panel of primary amines, not just arylalkylamines such as tryptamines and catecholamines, but also polyamines such as spermidine and

166 putrescine. In this context, a large percentage of AANATs studied function solely as N- acetyltransferases, rather than the putative fatty N-acyltransferases claimed to exist.

6.3 Long-chain acyltransferase activity in AANATs

Two exceptions were discovered however, namely D. melanogaster AANATL2

(DmAANATL2),58 and B. mori AANAT (Bm-iAANAT).24 While these enzymes were found to function with acetyl-CoA, activity was also recorded for long-chain fatty acyl-CoAs. Both enzymes were found to function with oleoyl-CoA and palmitoyl-CoA acting as acyl donor, and additionally for Bm-iAANAT, arachidonyl-CoA, myristoyl-CoA, and lauroyl-CoA.

Table 6.1: Summary of kinetic data obtained for Bm-iAANAT and DmAANATL2 utilizing acyl-CoAs of varying chain-length

-1 -1 -1 Bm-iAANAT KM (μM) kcat (s ) kcat/KM (M s ) Arachidonyl-CoA 1.2 ± 0.67 0.27 ± 0.02 (2.4 ± 1.3) x 105 Oleoyl-CoA 1.7 ± 0.51 0.42 ± 0.02 (2.4 ± 0.7) x 105 Palmitoyl-CoA 1.1 ± 0.3 0.51 ± 0.01 (4.9 ± 1.4) x 105 Myristoyl-CoA 0.92 ± 0.20 0.66 ± 0.01 (7.2 ± 1.5) x 105 Lauroyl-CoA 0.97 ± 0.12 1.3 ± 0.12 (1.4 ± 0.2) x 105 Acetyl-CoA 0.31 0.12 3.9 x 103 -1 -1 -1 DmAANATL2 KM (μM) kcat (s ) kcat/KM (M s ) Oleoyl-CoA 3.6 ± 0.58 0.075 ± 0.003 (2.1 ± 0.09) x 104 Palmitoyl-CoA 9.9 ± 1.6 0.16 ± 0.009 (1.6 ± 0.09) x 104 Butyryl-CoA 15 ± 1.8 0.53 ± 0.011 (2.9 ± 0.06) x 105 Acetyl-CoA 25 ± 1.8 1.3 ± 0.012 (2.2 ± 0.02) x 105

These data suggest that an AANAT or AANAT-like enzyme is indeed capable of synthesizing fatty acid amides, albeit at a very slow rate; recorded DmAANATL2 kcat for oleoyl-CoA is approximately 17-fold lower than that recorded for acetyl-CoA. The significantly decreased rate that comes with increasing acyl-chain length can be attributed to the potency of these fatty acid amides.19 Simply put, they are not needed as abundantly as their acetylated counterparts. It is interesting to note, however, that Bm-iAANAT is

167 essentially non-discriminatory in its acyl-chain length preferences. While functioning better with long-chain acyl-CoA donors, it shows relatively uniform activity from lauroyl-

(C12) to arachidonyl-CoA (C20). This is in contrast to DmAANATL2, which seems to balance a plummeting kcat with increased affinity (decreasing KM). These balance out quite comfortably in the measured specificity constants (kcat/KM) with a single order of magnitude difference between activity recorded for short- and long-chain acyl CoAs.

To date, Bm-iAANAT and DmAANATL2 are the only AANATs that have been identified that use long-chain acyl-CoAs as donor substrates. However, evidence for this common ancestor is apparent across many characterized AANATs. Firstly, comparison of kinetic constants and substrate specificity shows that the acyl-binding pocket of all

AANATs is fully capable of binding both short- and long-chain acyl CoAs. Long-chain acyl-CoAs have commonly been shown to be potent inhibitors for AANAT, rudimentarily

49,59 demonstrating nanomolar KI values. Ferry et al. also demonstrated sub-micromolar

60 inhibition for acyl-CoAs C10 and above against human AANAT. Additionally, a steadily decreasing KI/IC50 demonstrates that not only do fatty acyl-CoAs act as inhibitors, but it appears that the longer the chain, the stronger the inhibition. This trend of proportionally tighter binding is similar to that shown for DmAANATL2 KM values, which has also been seen in several other insect AANATs, such as Tribolium castaneum AANAT discussed in

Chapter 3 and Chapter 4.

168

Table 6.2: Summary of kinetic data obtained for TcAANAT0 and TcAANAT1b utilizing acyl-CoAs of varying chain-length

-1 -1 -1 TcAANAT0 KM (μM) kcat (s ) kcat/KM (M s ) Decanoyl-CoA 0.9 ± 0.2 0.8 ± 0.1 (9.6 ± 2.2) x 105 Butyryl-CoA 15 ± 1.8 5.5 ± 0.1 (3.7±0.5) x 105 Acetyl-CoA 25 ± 1.8 29 ± 0.6 (1.2±0.1) x 106 -1 -1 -1 TcAANAT1b KM (μM) kcat (s ) kcat/KM (M s ) Decanoyl-CoA 6.7 ± 0.8 3.4 ± 0.1 (5.1 ± 0.6) x 105 Butyryl-CoA 45 ± 4.9 14 ± 0.5 (3.1 ± 0.4) x 105 Acetyl-CoA 65 ± 9.8 52 ± 2.5 (8.0 ± 0.1) x 105

The implication of these trends is apparent with further comparison with a panel of other AANATs studied, such as DmAANATA,43 DmAgmNAT,42 and Bm-iAANAT3.49

Rather than displaying similar trends to T. castaneum AANAT, these display either slight preference for medium-chain acyl-CoA substrate binding, or no apparent preference.

Table 6.3: Summary of kinetic data obtained for DmAANATA, Bm-iAANATL3 and DmAgmNAT utilizing acyl-CoAs of varying chain-length

-1 -1 -1 DmAANATA KM (μM) kcat (s ) kcat/KM (M s ) Decanoyl-CoA 220 ± 60 0.04 ± 0.01 (1.7 ± 0.5) x 102 Octanoyl-CoA 18 ± 3 0.26 ± 0.01 (1.4 ± 0.3) x 104 Hexanoyl-CoA 23 ± 3 1.6 ± 0.1 (6.8 ± 0.9) x 104 Butyryl-CoA 36 ± 2 9.9 ± 0.4 (2.8±0.2) x 105 Acetyl-CoA 39 ± 12 16 ± 1 (4.1±1.3) x 105 -1 -1 -1 Bm-iAANAT3 KM (μM) kcat (s ) kcat/KM (M s ) Decanoyl-CoA 12 ± 5.5 0.05 ± 0.004 (4.2 ± 1.9) x 104 Octanoyl-CoA 10 ± 1.0 0.36 ± 0.005 (3.4 ± 0.3) x 104 Hexanoyl-CoA 13 ± 0.5 1.3 ± 0.01 (9.5 ± 0.4) x 104 Butyryl-CoA 14 ± 0.8 4.0 ± 0.04 (2.9±0.2) x 105 Acetyl-CoA 90 ± 3.6 64 ± 0.7 (7.1±0.3) x 105 -1 -1 -1 DmAgmNAT KM (μM) kcat (s ) kcat/KM (M s ) Decanoyl-CoA 70 ± 10 0.16 ± 0.01 (2.2 ± 0.3) x 103 Octanoyl-CoA 140 ± 10 0.61 ± 0.02 (4.4 ± 0.4) x 103 Hexanoyl-CoA 110 ± 3 1.1 ± 0.04 (9.7 ± 0.6) x 103 Butyryl-CoA 40 ± 6 3.3 ± 0.1 (8.0 ± 1) x 104 Acetyl-CoA 100 ± 6 23 ± 1 (2.2 ± 0.2) x 105

169

This indicates that rather than discrete AANAT families that can either long-chain

acylate or short-chain acylate, a single group of enzymes have evolved from a common

ancestor to accommodate both functions across a spectrum of specificity. Through

evolutionary stress, each insect has developed a panel of AANATs which stretches this

spectrum in the context of its biological requirements (Figure 6.1) In this sense, this family

should most accurately be called the arylalkylamine N-acyltransferases.

Long-chain acylation Short-chain acylation

DmAANATA Bm-iAANAT3 TcAANAT0/1 Bm-iAANAT

DmAgmNAT DmAANATL2

Figure 6.1: Schematic demonstrating the inherent spectrum of acylation utilized by various insect AANATs based off their obtained kinetic data. The further left the enzyme is found, the less likely it is to perform long-chain acylation.

6.4 Phylogenetic and Structural insight into long-chain acylation

It may be assumed that the divergence between long- and short-chain acylators in

the AANAT family can be traced phylogenetically. Construction of a Maximum Likelihood

tree in MEGA7,62,63 however, displays little in the way of trends in support of clear genetic

divergence one way or another (Figure 6.2). While the low sample size means accurate

hereditary examination is a primary concern, another trait that many members of the

AANAT family is their strikingly low sequence homology; often <40% from AANAT to

AANAT.64 This lack of sequence identity means that no definitive motifs can be attributed

to long- or short-chain acylation.

170

Figure 6.2: Phylogenetic tree built using the Maximum Likelihood method of AANATs from Drosophila melanogaster (Dm), Aedes aegypti (Aa), Tribolium castaneum (Tc) Periplaneta americana (Pa), Antheraea pernyi (Ap), Bombyx mori (Bm), Gallus gallus (Gg), Homo sapiens (Hs), Callorhinchus milli (Cm), and Danio rerio (Dr). AANATs known to favor long-chain acylation are boxed. Evolutionary analyses were conducted in MEGA7. The percentage of trees in which the associated taxa clustered together is shown next to the branches. 171

However, through the use of homology modeling, we are able to make predictions structurally as to why some AANATs can catalyze long-chain acylation, and some cannot.

A homology model for DmAANATL2 was built using the crystal structure of DmAANATA as the template.61 The high structural homology synonymous with the GNAT superfamily means that homology models are predicted to be very close to actual crystal structure data. Comparison of the homology model of DmAANATL2 with DmAANATA demonstrates, predictably, a relatively conserved structure. There are stark differences however, in the organization of the amine binding pocket. The basic shape of AANAT constitutes a cone, with the acyl-CoA donor stretching into the core of the enzyme, and the acceptor entering from the other side. The differences between the amine binding pockets are illustrated in Figure 6.3. An alignment of DmAANATA (blue) with

DmAANATL2 (red) shows that the opening for the amine, and thus, the active site, is much more disordered in DmAANATL2 (disorder shown in orange) than in DmAANATA

(disorder shown in cyan).

172

Figure 6.3: Overlay of crystal structure obtained for DmAANATA (3TE4 - blue) with homology model of DmAANATL2 (red). Shown in cyan is one region of disorder in the amine binding pocket of DmAANATA. Shown in orange is the much more pronounced region of disorder of the pocket of DmAANATL2.

The motif stretching from residue 94 to 104 of DmAANATA, shown in cyan in

Figure 6.3 corresponds to the disordered region in DmAANATL2 stretching from residue

78 to 91. This cyan region ends in a short α-helix, a feature missing from DmAANATL2.

Every other region displayed in orange is completely ordered in DmAANATA and, all together, this totals approximately 12% more disorder in DmAANATL2, all localized in the amine binding pocket. The implications of this in relation to the ability to catalyze long- chain acylation lies in the degrees of freedom offered by this disorder. A more dynamic amine binding pocket means that even if the acyl-CoA chain extends into the amine

173 pocket, the flexible nature of the pocket can more readily accommodate this and adjust to facilitate binding of the amine.

6.5 Conclusion

The identification of several panels of long-chain fatty acid amides in many different organisms and a distinct lack of understanding of many of their biosynthetic pathways is an intriguing conundrum. Kinetic analysis has shown us that AANATs in insects exist which can catalyze the reaction necessary. Through steric modification of the active site, insects have developed a way to fine tune the production of long- and short-chain fatty acid amides. By understanding this process more, it may be possible to shine light on the undetermined biosynthetic pathways behind many fatty acid amides in our bodies. It is apparent that AANATs carry the tools necessary to perform such a role.

Falcón et al. hypothesized that vertebrate AANAT (VTAANAT) and non-vertebrate

(NVTAANAT) diverged from each other 500 million years ago with the onset of AANAT neofunctionalization.65 Through understanding this process, we may be able to address the slew of diseases associated with the metabolism of these compounds such as

Alzheimer’s disease,66 Huntington’s disease,67 and Parkinson’s disease.68

While much work has been done to characterize insect AANATs, their heterogeneity means that each individual form of the enzyme holds with it a treasure trove of information pertaining to iAANAT evolution and fatty-acid amide metabolism. It is my hope that this text will enrich this field and facilitate proposition of iAANATs as potential insecticide targets.

174

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APPENDIX B:

SUPPELEMENTARY INFORMATION

S1: TcAANAT1b Michaelis Menten Plots

Figure S1: 5 mM histamine and varied acetyl-CoA

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Figure S2: 5 mM histamine and varied butyryl-CoA

Figure S3: 5 mM histamine and varied decanoyl-CoA

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Figure S4: 200 μM acetyl-CoA and varied dopamine

Figure S5: 200 μM acetyl-CoA and varied 2-2,-difluoro-2-phenethylamine

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Figure S6: 200 μM acetyl-CoA and varied histamine

Figure S7: 200 μM acetyl-CoA and varied norepinephrine

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Figure S8: 200 μM acetyl-CoA and varied octopamine

Figure S9: 200 μM acetyl-CoA and varied serotonin

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S2: TcAANAT0 (wild-type) Michaelis Menten Plots

Figure S10: 5 mM tryptamine and varied acetyl-CoA

Figure S11: 5 mM tryptamine and varied propionyl-CoA

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Figure S12: 5 mM tryptamine and varied butyryl-CoA

Figure S13: 5 mM tryptamine and varied crotonyl-CoA

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Figure S14: 5 mM tryptamine and varied DL-β-hydroxybutyryl-CoA

Figure S15: 5 mM tryptamine and varied decanoyl-CoA

200

Figure S16: 5 mM tryptamine and varied succinyl-CoA

Figure S17: 5 mM tryptamine and varied benzoyl-CoA

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Figure S18: 250 μM acetyl-CoA and varied dopamine

Figure S19: 250 μM acetyl-CoA and varied serotonin

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Figure S20: 250 μM acetyl-CoA and varied tryptamine

Figure S21: 250 μM acetyl-CoA and varied norepinephrine

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Figure S22: 250 μM acetyl-CoA and varied 2-phenethylamine

Figure S23: 250 μM acetyl-CoA and varied agmatine

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Figure S24: 250 μM acetyl-CoA and varied histamine

Figure S25: 250 μM acetyl-CoA and varied ethanolamine

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Figure S26: 250 μM acetyl-CoA and varied tyramine

Figure S27: 250 μM acetyl-CoA and varied octopamine

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S3: TcAANAT0 (Mutants) Michaelis Menten Plots

Figure S28: E25A – 5 mM tryptamine and varied acetyl-CoA

Figure S29: E25A – 250 μM acetyl-CoA and varied tryptamine

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Figure S30: E25D – 5 mM tryptamine and varied acetyl-CoA

Figure S31: E25D – 250 μM acetyl-CoA and varied tryptamine

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Figure S32: R134A – 5 mM tryptamine and varied acetyl-CoA

Figure S33: R134A – 250 μM acetyl-CoA and varied tryptamine

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S4: Bm-iAANAT3 Michaelis Menten Plots

Figure S34: 8 mM tryptamine and varied acetyl-CoA

Figure S35: 8 mM tryptamine and varied butyryl-CoA

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Figure S36: 8 mM tryptamine and varied hexanoyl-CoA

Figure S37: 8 mM tryptamine and varied decanoyl-CoA

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Figure S38: 500 μM acetyl-CoA and varied dopamine

Figure S39: 500 μM acetyl-CoA and varied histamine

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Figure S40: 500 μM acetyl-CoA and varied norepinephrine

Figure S41: 500 μM acetyl-CoA and varied octopamine

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Figure S42: 500 μM acetyl-CoA and varied serotonin

Figure S43: 500 μM acetyl-CoA and varied tryptamine

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Figure S44: 500 μM acetyl-CoA and varied tyramine

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S5: SDS Gels

70 70

35 35 25

15 15

Figure S45: SDS-PAGE gel of purified TcAANAT1b (left), TcAANAT0 (center), and Bm-iAANAT3 (right). Lane 1 – PageRuler™ prestained protein ladder (Thermo Scientific), Lane 2 – Purified protein

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