Purification and Characterization of Rhodobacter Sphaeroides 2.4.1 Hemt and Comparison with Hema Isoenzyme

Home , Bacteriochlorophyll, Cobalamin biosynthesis, Corrinoid, Rhodobacter, Rhodobacter capsulatus, Rhodobacter sphaeroides

Purification and Characterization of Rhodobacter sphaeroides 2.4.1 HemT and Comparison with HemA Isoenzyme

James Kaganjo

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2013

Committee:

Jill Zeilstra-Ryalls, Ph.D, Advisor

Raymond Larsen, Ph.D

Scott Rogers. Ph.D

James Kaganjo

iii ABSTRACT

Jill Zeilstra-Ryalls, Advisor

Rhodobacter sphaeroides synthesizes heme, vitamin B12 and bacteriochlorophyll to support its metabolic versatility that includes the ability to obtain energy by chemo- and phototrophy. The common precursor of all tetrapyrroles is 5-aminolevulinic acid

(ALA) which in these bacteria is formed from the condensation of glycine and succinyl-

CoA, a reaction catalyzed by PLP-dependent ALA synthase. ALA synthase is a member of the class II of fold type I PLP-dependent enzymes that also includes 2-amino-3- ketobutyrate-CoA ligase (KBL), serine palmitoyltransferase (SPT) and 8-amino-7- oxononanoate synthase (AONS). In R. sphaeroides strain 2.4.1, there are two ALA synthase genes, hemA and hemT, and when expressed each one alone can satisfy the requirement for ALA under all conditions that have been examined. In order to understand the role of the two ALA synthase isoenzymes, purification, characterization and comparison of the characteristics of both isoenzymes is necessary.

In this study, recombinant polyhistidine-tagged HemT (rHemT) was purified using nickel affinity chromatography, and it was characterized in terms of optimum pH and temperature, the effect of added hemin, its kinetic properties, its secondary structure, the presence of disulfide bonds, and its activity using an alternative substrate. The properties of rHemT were then compared to those of recombinant polyhistidine-tagged

HemA (rHemA), which was analyzed separately. The specific activity of rHemT was more than 10-fold lower than that of rHemA. The glycine Km for both proteins was the same, although the catalytic turnover rate for glycine was more than 10-fold higher for

iv rHemA than for rHemT. The succinyl-CoA Km for rHemA was 2-fold lower than for rHemT, and again the catalytic turnover was 10-fold higher for rHemA than for rHemT.

These differences indicate that HemT is a low activity ALA synthase when compared to

HemA, which would be explained if HemT has a greater preference for substrates other than succinyl-CoA. Towards this end, the ability of rHemT to use acetyl-CoA was evaluated. Using purified rHemT no detectable product was formed, indicating that succinyl-CoA is the preferred substrate.

Together with the comparisons of the HemA and HemT characteristics, a consideration of the substrates used in ALA production has suggested a possible explanation for the need to have both enzymes. This relies on the hypotheses that, (1) when hemA and hemT are both expressed, the products are able to form heterodimeric proteins, and (2) such heterodimers are less active than HemA homodimers, although possibly more active than HemT homodimers; i.e. hemT behaves in a dominant negative fashion to hemA. If true, then the role of HemT could be to reduce ALA synthase activity in the cell when succinyl-CoA and/or glycine are required for energy metabolism and protein synthesis, respectively.

v ACKNOWLEDGMENTS

I would like to thank Dr. Zeilstra-Ryalls for her immense guidance and mentorship for the last two years and also for giving me the opportunity to work in her laboratory. I would also like to express my gratitude to the members of my committee

Dr. S. Rogers and Dr. R. Larsen for their understanding and support during my study.

To Dr. M. Suwansaard (Joy), I am forever indebted for her guidance and mentorship when I joined the lab and throughout my master’s study. To all our lab members past and current am grateful for their helpful comments and ideas. Finally I would like to acknowledge my friends and family for their support and guidance.

TABLE OF CONTENTS

Page

CHAPTER 1. BACKGROUND INFORMATION AND SPECIFIC AIMS ...... 1

INTRODUCTION ...... 1

Rhodobacter sphaeroides...... 1

Tetrapyrroles ...... 2

ALA biosynthesis...... 4

ALA synthase isoenzymes ...... 7

SPECIFIC AIMS ...... 8

CHAPTER 2. PURIFICATION AND CHARACTERIZATION OF ACTIVE

POLYHISTIDINE-TAGGED HemT ENZYME ...... 10

INTRODUCTION … ...... 10

MATERIALS AND METHODS ...... 10

Bacterial strains, plasmids and growth conditions ...... 10

rHemT affinity purification ...... 12

Determination of rHemT protein concentration ...... 13

ALA synthase and 2-amino-3-ketobutyrate CoA ligase (KBL)

activity assays ...... 14

pH and temperature profile determinations ...... 15

Effect of added hemin ...... 15

Kinetic constant determinations ...... 15

Disulfide bond analysis ...... 16

Treatment with 4-acetamido-4’-maleimidylstilbene-2,2’- vii

disulfonic acid ...... 16

Treatment with 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole ...... 16

Circular dichroism spectroscopy...... 17

SDS-PAGE ...... 18

Immunoblot analysis ...... 18

InVision™ His-tag In-gel Stain assay ...... 19

Protein modeling and bioinformatic analysis ...... 19

Chemicals, reagents, and antisera ...... 19

RESULTS ...... 20

Expression and purification of rHemT ...... 20

SDS-PAGE, immunoblot analysis and InVisionTM His-tag analysis...... 21

Protein concentration determination ...... 22

Optimum Temperature and pH ...... 23

Effect of added hemin ...... 23

Glycine and Succinyl-CoA Km determinations ...... 24

Is HemT an ALA synthase or a KBL? ...... 25

Circular dichroism (CD) analysis of rHemT secondary structure ...... 27

Disulfide bond determination ...... 29

DISCUSSION ...... 31

CHAPTER 3. COMPARISON OF R. sphaeroides 2.4.1. POLYHISTIDINE-TAGGED HemA

WITH POLYHISTIDINE-TAGGED HemT ...... 32

INTRODUCTION ...... 32

PROPERTIES OF PURIFIED R. sphaeroides 2.4.1 rHemA versus rHemT ...... 32 viii

DISCUSSION ...... 36

REFERENCES ...... 38 ix

LIST OF FIGURES/TABLES

Figures Page

1 Diagram showing the basic structure of tetrapyrroles ...... 2

2 Schematic diagram of tetrapyrrole biosynthesis in Rhodobacter sphaeroides ...... 4

3 The two pathways of 5-aminolevulinic acid biosynthesis ...... 5

4 Schematic representation of the plasmid map of the expression vector pIND4-hemT 11

5 The amount of ALA formed per mg of protein versus assay incubation time ...... 21

6 Immunoblot and His-tag staining of rHemT...... 22

7 Effect of pH and temperature on rHemT activity ...... 23

8 Effect of added hemin on rHemT activity ...... 24

9 Substrate concentration versus initial velocity (Vo) and non-linear regression

plots for the determination of glycine and succinyl-CoA Kms ...... 25

10 Tertiary structure superposition of 3-D models R. sphaeroides 2.4.1

rHemT and KBL ...... 27

11 Circular dichroism spectra of rHemT in the far UV region (200–250 nm) ...... 29

12 SDS-PAGE gel of rHemT treated with AMS ...... 30

13 Amino acid squence alignment of wild type HemT and HemA, together with

the R. capsulatus ALA synthase (Rcaps)...... 33

14 Tertiary structure model superpositions of R. sphaeroides 2.4.1 ALA synthases

and Rhodobacter capsulatus ALA synthase (13)...... 34 x

Tables

1 Purification profile of rHemT ...... 20

2 Kinetic parameters determined for rHemT ...... 25

3 Specific enzyme activity of polyhistidine-tagged HemT using glycine with

succinyl-CoA versus glycine with acetyl-CoA ...... 27

4 Theoretical and calculated secondary structure values of rHemT ...... 28

5 Comparison of the kinetic properties of Rhodobacter sphaeroides 2.4.1

rHemA and rHemT...... 35 xi

LIST OF ABBREVIATIONS

3-D 3-dimensional

ALA 5-aminolevulinic acid

ALAS 5-aminolevulinic acid synthase

ATP Adenosine-5’-triphosphate

AMS 4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid

BSA Bovine serum albumin

C-C Carbon-Carbon

CD Circular dichroism

CoA Coenzyme A

DHAPS 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase

DHAP 3-Deoxy-D-arabino-heptulosonate-7-phosphate

DMSO Dimethyl sulfoxide

DTT Dithiothreitol

GLU glutamate

GSAM glutamyl-1-semialdehyde aminotransferase

IPTG isopropyl β-D-1-thiogalactopyronoside

Kb Kilobase

KBL 2-amino-3-ketobutyrate-CoA ligase kDa Kilo Dalton

LOMETS Local Meta-Threading-Server

Mb Megabase mg milligram xii

mM millimolar

NBD-Cl 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole

nm nanometer

OD optical density

PLP pyridoxal-5-phosphate

rHemA recombinant polyhistidine-tagged HemA

rHemT recombinant polyhistidine-tagged HemT

RNA Ribonucleic acid

S Substrate

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TCA Trichloroacetic acid tRNA transfer RNA tRNAGLU glutamate transfer RNA

g microgram

l microliter

M micromolar

UV Ultra violet

Vmax maximum velocity

Vo initial velocity

CHAPTER 1: BACKGROUND INFORMATION AND SPECIFIC AIMS

INTRODUCTION

Rhodobacter sphaeroides

Rhodobacter are sphaeroides gram-negative purple non-sulfur bacterium belonging to the

alpha-3 subclass of Proteobacteria. These organisms possess a wide range of metabolic

capabilities that include aerobic and anaerobic respiration, anoxygenic photosynthesis, and

fermentation (reviewed in 34). They are also little constrained by nutritional needs as they can

fix both atmospheric nitrogen and carbon and so require nothing more than mineral salts and a

few vitamins. Such metabolic fortitude involves the regulated synthesis of a multitude of

proteins and also other molecules such as tetrapyrroles enabling the cells to perform the variety

of different metabolic processes (reviewed in 34, 27 and 52).

In addition to its metabolic complexity, R. sphaeroides also possess high degrees of

genome complexity. In fact, this was the first bacterium found to have two chromosomes, and it became necessary to consider what criteria would define it as a chromosome, as opposed to a

plasmid. Deemed acceptable were the presences of ribosomal RNA operons, together with

essential genes such as glyceraldehyde-3-phosphate dehydrogenase (43). In strain 2.4.1, whose

genome was the first to be sequenced, chromosome I is 3.2 Mb and chromosome II is 0.9 Mb. In

addition, it has 5 circular endogenous plasmids. Four of the plasmids are approximately 0.1 Mb

in size, while the fifth plasmid is approximately 37 kb. Gene duplications are prevalent in R.

sphaeroides genomes. In some cases, homologues are both present on one chromosome, while

in others copies are distributed over both chromosomes (reviewed in 26). Among these

duplicates are the hemA and hemT genes that encode ALA synthases, the first enzyme in 2

tetrapyrrole biosynthesis. The presence of duplicate genes that may be differentially regulated

and expressed has the potential to increase the ability of the organism to adapt to environmental

changes, increasing the survival of the organism (reviewed in 26).

Tetrapyrroles

Tetrapyrroles are porphyrin compounds, consisting of four pyrrole rings that are joined together by methene bridges. Because they can bind metal ions, an apt description of these molecules is that they are organic packages for metal ions that perform important biological functions (Fig. 1). Tetrapyrroles are essential, and could be considered as “the key to life” based on their critical roles in catabolic and anabolic metabolisms.

1 2

N N

4 3

Figure 1. Diagram showing the basic structure of tetrapyrroles. The four pyrrole rings, numbered 1-4 are joined by methene bridges. The box at the center of the ring structure labeled “X” indicates the position at which iron, magnesium or cobalt metal ions are bound in the case of heme, bacteriochlorophyll and vitamin B12, respectively; all are present in R. sphaeroides.

Rhodobacter sphaeroides synthesizes three types of tetrapyrroles; the iron porphyrin

heme, the magnesium porphyrin bacteriochlorophyll, and the cobalt porphyrin vitamin B12.

These are all produced via a branching biosynthetic pathway (Fig. 2; reviewed in 44, 48 and 51), which begins with the formation of the common precursor molecule 5-aminolevulinic acid

(ALA; reviewed in 4). Both heme and bacteriochlorophyll participate in energy production processes. Heme bound to cytochromes is required for electron transport associated with both chemotrophy and phototrophy (reviewed in 4). Unlike heme, the role of bacteriochlorophyll in 3

these bacteria as a component of both the light harvesting complexes and the photosynthetic

reaction center is limited to phototrophic energy production. The corrinoid cobalamin vitamin

B12 functions as a cofactor for enzymes involved in hydrogen atom transfer reactions, serving as the source of the free radical that makes possible catalysis of reactions on molecules that are otherwise unreactive (3). It is interesting to note that, among the vitamin B12-dependent enzymes

is magnesium-protoporphyrin monoethyl ester cyclase, which participates in the synthesis of

bacteriochlorophyll (reviewed in 48).

A balanced ratio of the tetrapyrroles in the cell is important to meet the overall metabolic

requirements for these compounds. The most dramatic changes in cellular tetrapyrrole content

are brought about by changes in oxygen availability. When oxygen is plentiful, the cells rely

upon aerobic respiration to obtain energy, and all genes specific for phototrophy are repressed,

including those of the tetrapyrrole biosynthesis branch leading to the formation of

bacteriochlorophyll. As oxygen tensions fall, in preparation for anoxygenic phototrophic energy

production, both heme and especially bacteriochlorophyll production increases. To

accommodate this ramping up of tetrapyrrole levels, tetapyrrole precursor biosynthesis must also

increase.

Glycine + Succinyl-CoA

ALA synthase

5-aminolevulinic acid (ALA)

Co2+ Uroporphyrinogen III Vitamin B12

Protoporphyrin IX

Fe2+ Mg2+

Heme Bacteriochlorophyll

Figure 2. Schematic diagram of tetrapyrrole biosynthesis in Rhodobacter sphaeroides. Heme, bacteriochlorophyll and cobalamin are synthesized in a branched pathway from the common precursor, 5- aminolevulinic acid (ALA). In this bacterium, ALA is formed from the condensation of glycine and succinyl-CoA catalyzed by ALA synthase.

ALA Biosynthesis

In plants, archaea and most bacteria ALA is formed via the C5 pathway. In this pathway

ALA is formed from the 5-carbon compound glutamate (reviewed in ref. 39). The first step in

this pathway is the formation of glutamyl-tRNAGLU, using the same tRNA and catalyzed by the same tRNA synthetase as is used in protein synthesis. The two remaining reactions are catalyzed by pathway-specific enzymes. First, glutamyl-tRNA reductase reduces glutamyl-tRNAGLU to glutamyl-1-semialdehyde and tRNAGLU. Then the pyridoxal phosphate (PLP)-dependent

glutamyl-1-semialdehyde aminotransferase (GSAM) adds an amino group to form ALA (Fig. 3).

Animals and α-proteobacteria, including R. sphaeroides, synthesize ALA via the C4 or

the so-called Shemin pathway, named after its discoverer (40). The pathway-specific event is the

condensation of the four-carbon skeleton of succinate in its activated form, succinyl-CoA, with

glycine which is catalyzed by the PLP-dependent enzyme ALA synthase (40). Thus, in

organisms using the C4 pathway ALA production is metabolically linked to both protein 5

synthesis via the glycine requirement, and also to central carbon metabolism through the requirement for succinyl-CoA.

Figure 3. The two pathways of 5-aminolevulinic acid biosynthesis. The C5 pathway, which is present in plants and most bacteria and forms ALA from glutamyl-tRNAGLU catalyzed by two pathway-specific enzymes (reviewed in 39), is at the top and the C4 (Shemin) pathway, which is present in all animals and the α-proteobacteria, is at the bottom and forms ALA from condensation of glycine and succinyl-CoA (40).

ALA synthases are homodimeric proteins, and each polypeptide has a molecular mass

ranging from 44-65 kDa. They belong to class II aminotransferases of the fold type I (aspartate

aminotransferase) family of PLP-dependent enzymes that also includes 8-amino-7- oxononanoate, serine palmitoyl transferase, 2-amino-3-ketobutyrate-CoA ligase and histidinol- phosphate aminotransferase. Enzymes belonging to this family catalyze the formation of a 2- 6

amino-3-ketoacid intermediate that is bound to the enzyme through the formation or cleavage of

a C-C bond between an amino acid and an acyl-CoA. The fold type I category of PLP-dependent

enzymes share a similar structure, even though they catalyze different reactions. Having a very

similar tertiary structure may be an indication that they belong to the same evolutionary lineage

(reviewed in 32 and 14).

The reaction mechanism of ALA synthases involves the displacement of the Schiff base

between the active site lysine residue of the enzyme and PLP (internal aldimine bond) by glycine

to form a second Schiff base between glycine and PLP (external aldimine bond) forming a

quinonoid intermediate (carbanion) which is formed through the loss of a proton at the alpha carbon of glycine. This carbanion then attacks succinyl-CoA, releasing ALA, CO2 and coenzyme A. The release of ALA from the enzyme is considered to be the rate-limiting step of the reaction. (54, 2, 29).

While in most organisms only one or the other of these pathways for ALA formation is present, interesting exceptions to this generality have been identified. In Euglena gracilis the C5 pathway is restricted to the chloroplasts (45). In the bacterium Streptomyces nodosus subsp. asukaensis, ALA for tetrapyrrole biosynthesis emanates solely via the C5 pathway, while the C4 pathway supplies ALA for asukamycin production in that organism (33). A hypothesis as to how this confounding dedicated use of the common metabolite is achieved is based upon an elegant study of events subsequent to ALA formation in the synthesis of asukamycin and other natural products that all contain a 2-amino-3-hydroxycylcopent-2-enone five membered rings, or C5N

units (55). In that study, it was found that ALA is converted to ALA-CoA by an acyl-CoA

ligase, which then serves as a substrate for a newly discovered second activity of the ALA

synthase, cyclizing the ALA-CoA to C5N. The hypothesis is, then, that the acyl-CoA ligase 7

activity is effective at making ALA unavailable for porphobilinogen synthase, which is the

second enzyme in tetrapyrrole biosynthesis, and so directs flow towards C5N rather than porphobilinogen synthesis.

ALA synthase isoenzymes

Isoenzymes are multiple forms of an enzyme that are similar in structure and catalyze the same reaction. It is thought that the presence of such apparent catalytic redundancy is explained by the potential to increase metabolic flexibility. This may be achieved by having enzymes with distinctive properties, which can be combined with differential expression of the respective genes.

A textbook example of the role of isoenzymes in the cell is that of the 3-deoxy-D- arabino-heptulosonate-7-phosphate synthases (DAHPS) in E. coli. DAHP is the common precursor to the three aromatic amino acids, and each of the three DAHPS enzymes are differentially feed-back regulated by tyrosine, phenylalanine, and tryptophan in order to achieve their balanced production in the cell (35).

In the tetrapyrrole biosynthetic pathway, isoenzymes of ALA synthase are present in both mammals and prokaryotes. In mammals, the differential expression of the two ALA synthase genes is well characterized. One of the genes is exclusively expressed in developing erythrocytes where the demand for ALA is higher than in other tissues, for the synthesis of heme required to form hemoglobin. The other gene is the so-called housekeeping gene, as it is constitutively expressed in all cells (37). While the literature is replete with studies of the erythropoetic enzyme, the housekeeping enzyme has been understudied. Therefore, differences in their properties are not yet defined. The first bacterium found to have two ALA synthase genes is R. sphaeroides, and studies have shown that those genes are also differentially expressed 8

(30 and 53). Because only one of the two R. sphaeroides gene products has been purified in active form, nothing is known about the relative properties of these two proteins. Until such time as the enzymes have been characterized, the full significance of having duplicate ALA synthase genes is not known.

SPECIFIC AIMS

Based on recent findings in the lab of Jill Zeilstra-Ryalls, a clearer picture regarding the differential expression of the two ALA synthase genes in R. sphaeroides, hemA and hemT, is emerging (12). The hemA gene is transcribed under all conditions, but it is upregulated in the absence of oxygen. The hemT gene is only expressed in certain strains of R. sphaeroides. This is thought to be explained by evidence suggesting that its transcription relies upon specialized sigma factors that are not present in all strains. The activity of the sigma factors is governed by growth conditions, and hemT transcription appears to be maximal when cells are relying upon anaerobic respiration for energy production. Since hemA expression is high under those same conditions, the cytoplasm would contain both HemA and HemT enzymes. This raises the question as to whether or not the enzymes possess unique properties that require them both to be expressed in this organism in order to support its full range of metabolic capabilities.

The central hypothesis of this thesis is that HemA and HemT may possess unique properties that may require them to be encoded by this organism. The purpose of this investigation is to test this hypothesis, and so address the question as to why R. sphaeroides has two ALA synthases.

The specific aims are:

1. Purify and characterize active HemT enzyme. 9

2. Compare the characteristics of HemT to those of HemA, which is currently under similar investigation. 10

CHAPTER 2: PURIFICATION AND CHARACTERIZATION OF ACTIVE

POLYHISTIDINE-TAGGED HemT ENZYME.

INTRODUCTION

HemT has remained uncharacterized until now. This is because previous efforts have not successfully produced soluble, active enzyme (6). Towards understanding the significance of two ALA synthase isoenzymes in R. sphaeroides, recombinant polyhistidine-tagged HemT

(rHemT) from R. sphaeroides 2.4.1 was purified and characterized. The properties that were characterized included specific activity, optimum pH and temperature, the effect of added hemin, activity with an alternative substrate, disulfide bond detection, and secondary structure analysis.

Characterization of these properties is necessary in order to be able to compare them with those of HemA, which has been analyzed separately (50), towards understanding why there are two isoenzymes of ALA synthase in R. sphaeroides.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. The expression system used for the production of recombinant polyhistidine-tagged HemT (rHemT) protein is composed of the host bacterium R. sphaeroides mutant strain AT1 and the plasmid pIND4-hemT (Fig. 4).

Rhodobacter sphaeroides mutant strain AT1 is derived from the wild type strain 2.4.1. Both the hemA and hemT genes are disabled, and hence the bacteria require ALA to grow (30). Plasmid pIND4-hemT was constructed by insertion of a hemT gene. The gene was first modified by the removal of its nonsense codon, replacing it with sequences that comprise a HindIII restriction site. Construction of pIND4-hemT then involved cloning using the NcoI site that is naturally 11

present at the 5' end of the coding sequences and the engineered HindIII site at the 3' end. This

creates a hemT gene coding for a protein having a C-terminus hexa-histidine tag that are added

from the pIND4 vector (16).

A 5 ml inoculum of a freshly grown preculture was used to inoculate 100 ml of Sistrom's

succinate minimal medium (42) supplemented with kanamycin (50 g/ml) for plasmid

maintenance. This preculture was grown to early stationary phase, having an optical density

(OD) at 660 nm of 0.8 – 1.0, and was then diluted into 1 liter of fresh medium. The culture was incubated until early stationary phase. Culture conditions were 30oC with shaking at 175 rpm in

a New Brunswick Scientific (Excella E25) incubator shaker (New Brunswick Scientific, Enfield,

CT). Because the hemT gene is expressed from a synthetic promoter that is regulated by the LacI

repressor, which is also coded for by the pIND4 plasmid, 0.4 mM isopropyl β-D-1-

thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO) was used to induce transcription

of the hemT gene. Inducer is required at all times to supply adequate ALA for growth of the

bacteria.

lacIq ColE1

pIND4‐hemT pMG160

PA1/04/03

hemT‐6 x his KnR

Figure 4. Schematic representation of the plasmid map of the expression vector, pIND4-hemT. The figure shows key features of the expression vector that is derived from plasmid pIND4 (16): The lacIq gene, which codes for the LacI repressor, is shown in blue, the synthetic LacI-regulated promoter is shown by a black arrow, the kanamycin resistance gene used for plasmid selection is shown in purple and the orange rectangle depicts the engineered hemT gene having sequences coding for a polyhistidine-tag that is in frame with the C-terminus. Also identified is the ColE1, origin of replication that allows for 12

replication of the plasmid in Escherichia coli and pMG160 sequences, an endogenous plasmid of Rhodobacter blasticus that can also replicate in Rhodobacter sphaeroides (17).

rHemT affinity purification. Crude lysates were prepared from cells harvested by

centrifugation at 3,836 x g, at 4oC for 15 min in a Beckman J2-21 centrifuge (Beckman Coulter,

Inc., Brea, CA) with a fiberlite F14BA-6x250Y rotor (Thermo Fisher Scientific Inc., Rockville,

MD). The pelleted cells were resuspended in 2 ml of lysis buffer (20 mM tricine, 10% glycerol,

15 mM NaCl, 5 mM imidazole, 20 µM pyridoxal 5’-phosphate (PLP), and 5 mM β- mercaptoethanol, pH 7.2) with 100 µl protease inhibitor complex that has a cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO) that target a wide range of proteases including serine, cysteine, aspartyl- and metallo-endopeptidase proteases that may be present in the bacteria. The cells were lysed by passaging them through a French pressure cell (Thermo Fisher Scientific

Inc., Milford, MA) at 700 lb/in2. The crude lysate was then incubated with DNase I (Sigma-

Aldrich, St. Louis, MO) for 30 min on ice to fragment DNA that otherwise blocks the flow of the

affinity purification resin. The insoluble material was then cleared from the lysate by

centrifugation at 20,817 x g at 4oC for 15 min using an Eppendorf 5810R refrigerated centrifuge and a F-45-30-11 fixed-angle rotor (Eppendorf North America, Hauppauge, NY). The rHemT protein was purified from the clarified crude lysate using nickel affinity chromatography, performed at 4oC with His60 Ni superflowTM resin (Clontech Laboratories Inc., Mountain View,

CA). Purification steps were (i) load the crude lysate onto the column by gravity feed, then

incubate the column for 30 minutes on a 3-D rotator (Labline Scientific Instruments, Mumbai,

India); (ii) clamp the column vertically and then remove unbound protein by washing the column

with wash buffer I (20 mM tricine, 10% glycerol, 150 mM NaCl, 10 mM imidazole, 20 µM PLP,

and 5 mM β-mercaptoethanol, pH 7.2) until the absorption at 280 nm is zero; (iii) remove

nonspecifically bound protein by washing the column with wash buffer II (20 mM tricine, 10% 13

glycerol, 25 mM NaCl, 40 mM imidazole, 20 µM PLP, and 5 mM β-mercaptoethanol, pH 7.2);

(iv) elute rHemT protein using elution buffer (20 mM tricine, 10% glycerol, 25 mM NaCl, 350 mM imidazole, 20 µM PLP, and 5 mM β-mercaptoethanol, pH 7.2). the eluted protein was stored in -20oC in elution buffer (20 mM tricine, 10% glycerol, 25 mM NaCl, 350 mM imidazole, 20 µM PLP, and 5 mM β-mercaptoethanol, pH 7.2).

Determination of rHemT protein concentration. A modification of the procedure described by Greenfield (15) was used to precisely determine the protein concentration. First, extinction coefficients are calculated, using the following formulas (15, 13):

288 = 4815(W) + 385(Y) + 75(C) (1)

280 = 5690(W) + 1280(Y) + 120(C) (2)

W, Y and C are the number of tryptophans, tyrosines and cystines, respectively, per mole of

protein. Since the number of disulfide bonds present in rHemT is not known, as per the method

described by Eldoch (13), the number of cystines present were assumed to be zero. Then the

purified protein was dialyzed against dialysis buffer (20 mM tricine, 10% glycerol, 25 mM NaCl,

50 mM imidazole, 20 µM PLP, pH 7.2) to remove -mercaptoethanol and reduce the imidazole

concentration. A spectral analysis was performed using dialyzed protein and a Hitachi U-2910

spectrophotometer (Hitachi High Technologies America Inc., Schaumburg, IL). Sample and

reference cuvettes containing equal amounts of 6 M guanidine-HCl, pH 6.5, were zeroed against

each other across the spectrum from 250 to 350 nm. Then 90 l of the dialysis buffer was added

to the reference cuvette, 90 l of dialyzed protein was added to the sample cuvette, and the

spectrum was recorded between 350 and 250 nm. Protein concentrations were calculated using

absorbances at 280 and 288 nm and the respective extinction coefficients. These values should

be approximately the same, provided the enzyme was fully unfolded (15). 14

The Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA) was also used

to determine protein concentrations according to the manufacturer's instructions. A standard

curve was generated using bovine serum albumin. These values were then calibrated according to the concentrations determined spectrophotometrically based upon the calculated 288 and 280 values. All calculations based upon protein concentrations used these recalibrated values.

ALA synthase and 2-amino-3-ketobutyrate CoA ligase (KBL) activity assays. ALA synthase activity was determined using the method of Burnham (7) as modified by Neidle and

Kaplan (30). To start the reaction, samples of protein were added to 10.2 l succinyl-CoA (100

M), 15 l PLP (0.28 M) and 35 l glycine mix (98 mM glycine, 9.8 mM MgCl2, and 20 mM tricine, pH 7.2). The final reaction volume was 102 l. As control, milli-Q water was used instead of enzyme solution. Reactions were incubated at 37oC for 10 min and then halted by the addition of 50 l 10% trichloroacetic acid (TCA). Enzyme saturation was determined by varying the amount of protein added to the mix. The amount of ALA formed during the reaction was determined by first converting it to a pyrrole compound (ALA-pyrrole; 2-methyl-3-acetyl-4-(3- propionic acid) pyrrole) through chemical condensation at 100oC with 5 l acetylacetone in 200

l 1 M acetate buffer pH 4.7 for 15 min. A volume of 350 l of freshly prepared modified

Ehrlich’s reagent (6.7 mM p-dimethyaminobenzaldehyde, 42 ml glacial acetic acid, and 8 ml

70% perchloric acid) was then added to convert the ALA-pyrrole into a purple colored ALA-

pyrrole complex that is formed when the ALA-pyrrole reacts with p-

dimethyaminobenzaldehyde. After a 20 min incubation at room temperature to fully allow for

color development, the absorption at 556 nm was determined using the Hitachi U-2910

spectrophotometer. Enzyme activity was expressed as µmoles of ALA formed per hour, 15

calculated using an extinction coefficient () for the ALA-pyrrole derivative of 68,000 M-1 cm-1

(7) according to the following equation:

(3)

KBL assays were performed in the same manner, except 100 M acetyl-CoA was used instead of succinyl-CoA in the assay.

pH and temperature profile determinations. To determine the pH optimum of rHemT,

ALA synthase activity assays of the purified protein were performed as a function of pH. A range of pHs were achieved by adjusting the pH of the glycine mixtures used in the reactions.

The temperature optimum of purified polyhistidine-tagged HemT was determined by performing

ALA synthase activity assays as a function of temperature. To do so, the reaction mixtures were incubated in water baths at various temperatures for ten minutes.

Effect of added hemin. The effect of added hemin on the ALA synthase activity of rHemT was examined by adding hemin to the ALA synthase reaction mix to a final concentration of 0.1 µM, 1 µM, 10 µM, 100 µM or 200 µM. Stock solutions of hemin were prepared by dissolving it in a solution of 0.01 M NaOH in 50% ethanol and 50% milli-Q water.

The reactions were performed at 37oC.

Kinetic constant determinations. Data for determining the Km for glycine were collected by assaying ALA synthase activity with different concentrations of glycine at a constant succinyl-CoA concentration of 100 µM. Data for determining the Km for succinyl-CoA

were collected by assaying ALA synthase activity with different concentrations of succinyl-CoA

at a constant glycine concentration of 100 mM. The Kms were then calculated by plotting

enzyme velocities versus substrate concentrations, and fitting the data to the equation Vo = (Vmax 16

[S])/ (Km + [S]) using nonlinear regression analysis with SigmaPlot software (Systat Software,

San Jose, CA). Vmax was determined from the graph and kcat was calculated by dividing Vmax with

enzyme concentration.

Disulfide bond analysis. Two methods were used to investigate the presence of

disulfide bonds in purified rHemT as modified from Lee C. et al (23).

(1) Treatment with 4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid (AMS): Any

disulfides in the native (active) protein were reduced by treating 300 l (0.4 mg/ml) of purified

rHemT protein with 50 mM dithiothreitol (DTT) at 37oC for 10 min. The protein was then

precipitated by the addition of 100 µl of a 10% solution of TCA and centrifuged at 20,817 x g at

4oC for 15 min (Eppendorf centrifuge 5810R, 30-place fixed-angle rotor F-45-30-11). The pellet was resuspended in AMS buffer (15 mM AMS, 20 mM tricine, 10% glycerol, 25 mM NaCl, pH

7.2) and incubated for 1 h at 37oC. To fully oxidize the native (active) protein, it was treated with 1 mM hydrogen peroxide for 10 min at 37 C. The oxidized protein was then processed in the same way as the reduced protein. Both treated and untreated protein samples were mixed with equal volumes of 2 x Laemmli buffer (20% glycerol (v/v), 2% SDS (w/v), 2.5%

Bromophenol Blue (w/v), 5% β-mercaptoethanol (v/v), Tris-base 0.125 mM; ref. 21), incubated

5 min at 100oC and electrophoresed through a 8-16% gradient gel. Control samples were AMS-

treated and untreated protein.

(2) Treatment with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl): A volume of 750 l

purified rHemT (0.3 mg/ml) in elution buffer was reduced with 50 mM DTT for 10 min at 37oC

and then treated with 24 l of 20 mM NBD-Cl dissolved in dimethyl sulfoxide (DMSO) for 2 h

at 37oC. Residual NBD-Cl reagent was removed by dialysis with elution buffer overnight. Then

240 l of dialyzed protein was mixed with 360 l 8.4 M guanidine hydrochloride to completely 17

unfold the protein. Spectra were recorded from 220 nm to 550 nm using a Hitachi U-2910

spectrophotometer after recording the baseline with 360 l 8.4 M guanidine hydrochloride. For

the oxidized protein analysis, rHemT was oxidized with 1 mM hydrogen peroxide and then

treated the same way as the reduced HemT. The data was graphed using Sigmaplot software

(Systat Software).

Circular dichroism spectroscopy. Purified rHemT was concentrated using Amicon

Ultra-15 centrifugal filter device with a molecular mass cutoff of 10,000 daltons (Millipore,

Bedford, MA). A volume of 15 ml of the eluate was loaded into the filter device and centrifuged

at 4,000 x g for 12 min (Eppendorf 5810R refrigerated centrifuge) to a final protein

concentration of 0.30-0.35 mg/ml, which represented a 10-fold concentration. The concentrated

protein was then dialyzed overnight against dialysis buffer (20 mM tricine, 10% glycerol, 25 mM

NaCl, pH 7.2) to remove imidazole, β-mercaptoethanol and free PLP. The dialyzed enzyme was

centrifuged for 1 min at 6,708 x g using a Minispin plus centrifuge (Eppendorf N. America, Inc.

Hauppange, N.Y) at room temperature to remove any insoluble particles. Absorption spectra in

the far-UV region (190-240 nm) were collected using a 0.1 cm cuvette and an Aviv 62DS

Circular Dichroism spectrometer (Aviv Associates, Inc., Lakewood N.J.) at 25oC. The spectra were recorded in triplicate to ensure that the sample was at equilibrium and the signal was stable.

To analyze the data, the spectra were first averaged and corrected for the blank spectrum (buffer without enzyme). The values were then plotted using SigmaPlot software with 2-D smoothing, using the running average algorithm to remove any sharp variations and eliminate noise. The fractional alpha helical (H) content, expressed as a percentage, was estimated using the following equations (11):

H = ([]222 – 3,000)/(-36,000 – 3,000) (4)

H = ([]208–4,000)/(-33,000– 4,000) (5)

[]222 and []208 are molar residue ellipticities at 222 and 208 nm, respectively. The molar residue ellipticities of beta sheet and random coil at 222 nm and 208 nm are 3000 and 4000, respectively.

The molar residue ellipticity of alpha helix at 222 is -36,000 and at 208 nm is -33,000 (11). The data were also evaluated for secondary structure using the dichroweb website database

(24,46,47) and the K2D program, which predicts the secondary structure of a protein using neural networks that have been trained on proteins of known secondary structure (1). Molar residue ellipticity ([]) values were calculated based on the mean amino acid residue molecular weight of rHemT according to the following equation (15):

[] = (Total amino acids – 1) / protein concentration (mg/ml) (6)

SDS-PAGE. SDS-PAGE was performed using routine methods (38) and either 12% gradient polyacrylamide tris-glycine precast gels (Invitrogen, Carlsbad, CA) for protein purification analysis or 8 - 16% gradient gels (Pierce, Rockford, IL) were used for disulfide bond determination. Protein samples were mixed with equal volumes of 2 x concentrated Laemmli buffer (20% glycerol (v/v), 2% SDS (w/v), 2.5% Bromophenol Blue (w/v), 5% β- mercaptoethanol (v/v), Tris-base 0.125 mM; ref. 21) and then boiled for 5 min at 100oC.

Precision Plus ProteinTM Kaleidoscope TM standards (Bio-Rad Laboratories) were used to estimate the molecular masses of proteins.

Immunoblot analysis. Immunoblot analysismed was by perfortransferring proteins that were first resolved by SDS-PAGE onto a nitrocellulose membrane, using standard transfer protocols (20). After blocking the membrane overnight with 5% bovine serum albumin (BSA) in tris-buffered saline (10 mM Tris-HCl, 150 mM NaCl, pH 8.0) with 0.05% Tween-20, the 19

membrane was probed with monospecific polyclonal rabbit anti-HemT antiserum as primary

antibody (6, 25) and with goat anti-rabbit IgG conjugated with alkaline phosphatase as secondary

antibody (Sigma-Aldrich, St. Louis, MO). The immunocomplexes were visualized using the

BCIP®/NBT Liquid Substrate System (Sigma-Aldrich).

InVision™ his-tag in-gel stain assay. To detect the histidine-tagged HemT, rHemT

samples were first resolved by SDS-PAGE. The gel was then fixed using 100 ml fixing solution

(40 ml methanol, 10 ml glacial acetic acid and 50 ml milli-Q water) for 1 h and then washed 2 times for 10 min each with milli-Q water. The gel was incubated overnight with the ready-to-use solution of InVisionTM His-tag In-gel stain (Invitrogen Life Technologies, Carlsbad, CA) and then washed twice for 10 min each with 100 ml 20 mM phosphate buffer pH 7.8. The stained gel was imaged immediately.

Protein modeling and bioinformatic analysis. The predicted structures of wild type and rHemT were generated using the Local Meta-Threading-Server (LOMETS; ref. 49).

Superimposed images of the tertiary structures of R. sphaeroides 2.4.1 wild type and recombinant polyhistidine-tagged HemA, HemT and Rhodobacter capsulatus HemA was done using TM-align (56). Multiple amino acid sequence alignments of wild type HemT and other

ALA synthases were generated using ClustalW (22), and displayed using the GeneDoc Multiple

Sequence Alignment Editor and Shading Utility (31).

Chemicals, reagents, and antisera. Unless otherwise indicated, all chemicals were at least of ACS grade, all fine chemicals and reagents were purchased from Sigma-Aldrich (St.

Louis, MO) and Invitrogen Corp. (Carlsbad, CA).

RESULTS

Expression and purification of rHemT. Since R. sphaeroides AT1 is able to grow in

the presence of IPTG and in the absence of ALA, the recombinant C-terminus polyhistidine–

tagged HemT (rHemT) protein is functional. To develop a protocol that would maximize yield and purity of the protein a series of small-scale experiments were performed, in which binding, washing, and elution conditions were varied. Quality and quantity were assessed by SDS-PAGE and ALA synthase activity assays. The final optimized protocol is described in the Materials and

Methods. Using this protocol, typically approximately 37 mg of rHemT was isolated from a culture of 1 liter of cells grown in the presence of 0.4 mM IPTG to an OD660 of 0.8-0.1. The purification profile is provided in Table 1.

Table 1. Purification profile of rHemT.

Enzyme Protein Specific activity Purification Yield Sample activity concentration (Ua/mg) fold % (µmol ALA/h) (mg/ml)

Crude lysate 1.1 ± 0.2 5.0 ± 0.12 0.22 ± 0.04 1 100

Flow through 0.45 ± 0.01 2.6 ± 0.08 0.2 ± 0.005 0.9 41

Wash I 0.2 ± 0.003 0.25 ± 0.02 0.8 ± 0.01 3.6 18

Wash II 0.44 ± 0.006 0.005 ± 0.004 91 ± 2 414 40

Elution 0.8 ± 0.1 0.037 ± 0.002 26 ± 4 118 73

aUnits are defined as 1 µmol ALA synthesized per hour at 37oC.

The enzyme activity was found to be unstable, and declined rapidly after only a few days

when stored at either -20oC or -80oC. However, concentrating the protein to 0.3 mg/ml increased

stability of the activity, which remained constant for protein stored at -20oC for up to one month.

To determine the linear range of the ALA synthase assay for purified rHemT, the

incubation time (at 37oC) of the assay was varied. As shown in Fig. 5, steady state was achieved

by approximately 5 min, and for incubations between 5 and 20 min duration the production of

ALA was linearly proportional to the incubation time. Based on this result, the incubation times

used for all assays was 10 min, unless otherwise indicated.

Figure 5. The amount of ALA formed per mg of protein versus assay incubation time. The assay was performed as described in the Materials and Methods using 100 M glycine and 100 M succinyl-CoA. The points are the averages of two assays, and the ranges are indicated by the error bars. The graph was generated using Sigmaplot software (Systat Software).

SDS-PAGE, immunoblot analysis and InVision his-tag analysis. The affinity-purified

protein migrated as a single band on an SDS-PAGE. To confirm the identity of the protein an

immunoblot was probed with mono-specific polyclonal rabbit anti-HemT antiserum. A protein

of the correct molecular mass was detected, confirming that the purified protein is rHemT. In 22

addition, using the in-gel InvisionTM stain, a protein of the same mass was detected, confirming

that the immunodectable purified protein was polyhistidine-tagged. These results are shown in

Fig. 6.

Figure 6. Immunoblot, and His-tag staining of rHemT. Lane 1: immunoblot probed with rabbit anti- HemT antiserum as primary antibody (6,25), lane 2: Coomassie-stained adjacent lane on the same SDS- PAGE gel, lane M: molecular mass marker (sizes are indicated), lane 3: Coomassie-stained adjacent lane on the same SDS-PAGE gel of Invision His-tag stain (Life Technologies), and lane 4: Invision His-tag stain (Life Technologies) of SDS-PAGE.

Protein concentration determination. The molar extinction coefficients, 280 and 288, of rHemT were calculated according to the procedure described in the Materials and Methods.

The values were 34,280 M-1 cm-1 and 22,725 M-1 cm-1, respectively. When the absorbances at

280 and 288 nm of a sample of purified protein were measured, the calculated protein

concentrations using the corresponding extinction coefficients were in close agreement, differing

by approximately 1% (1 g/ml).

The protein concentration determinations using the Bio-Rad protein assay relies upon

Coomassie blue dye binding to basic and aromatic amino acid residues in a protein. The protein

concentration is then calculated from a standard curve generated using bovine serum albumin

(BSA). Therefore, it is a relative, rather than absolute value since the basic and aromatic amino

acid composition is not the same in every protein, and can differ from BSA. While this method

is unsuitable for circular dichroism analysis of secondary structure, which requires a precise

measure of protein concentration, the Bio-Rad assay is more sensitive than spectroscopic 23

determinations, and lends itself well to rapid measurements of many samples. To report absolute

protein concentrations, values determined using the Bio-Rad assay were adjusted by a factor of

1.2, based upon a comparison of concentration determined using both methods.

Optimum temperature and pH. The effect of temperature on the specific activity of

rHemT was determined by incubating the enzyme reaction mixture of pH 7.2 at different

temperatures. To examine the influence of pH on enzyme activity, the pH of the reaction

mixture was adjusted, and the reaction was incubated at 37oC . Using these conditions, the

optimum temperature was 37-42oC (Fig. 7A), and the optimum pH was 7.2 (Fig. 7B).

Figure 7. Effect of pH and temperature on rHemT activity. Shown are percent activity versus pH (A) and temperature (B). The y-axes have been truncated to provide maximum visual resolution of the values.

Hemin inhibition. An early studyhase of ALA activity synt purified from R. sphaeroides reported the activity is inhibited by hemin (8). Hemin is one of the end products of the tetrapyrrole biosynthetic pathway, and so this would represent regulation by a feed-back inhibition mechanism. However the study preceded the discovery of two ALA synthase genes, and since expression of hemA and hemT has not been examined in the strain used, the molecular composition of the purified enzyme is not known. To investigate whether rHemT is inhibited by hemin, specific activities were determined in the presence of varying concentrations (0-200 M)

of hemin (Fig. 8). In the presence of 200 M hemin, specific activity was reduced by 24

approximately 35%. The same concentration of hemin resulted in 87% inhibition of the ALA

synthase activity reported by Burnham and Lascelles (8). This difference could be explained by

the possibility that HemA protein comprises the majority of the activity, and that HemA enzyme

is feedback inhibited by hemin.

Figure 8. Effect of added hemin on rHemT activity. Shown is percent activity versus hemin concentration. The y-axis has been truncated in order to provide maximum visual resolution of the values.

Glycine and succinyl-CoA Km determinations. The Kms for glycine and succinyl-CoA were determined by non-linear regression of the plots of substrate concentration versus initial velocities of each substrate using the equation Vo = (Vmax [S]) / (Km + [S]) (Sigmaplot). The Km

for glycine was determined from the plot of initial velocities measured over a range of glycine

concentrations from 0-150 M at a constant succinyl-CoA concentration of 100 M (Fig. 9A).

The Km for succinyl-CoA was determined from the plot of initial velocities measured over a range of succinyl-CoA concentrations from 5 M-100 M at a constant glycine concentration of

100 M (Fig. 9B). The Vmax, kcat, and kcat/Km values were also deduced from the plots. The

values for all of these kinetic parameters are provided in Table 2. 25

Figure 9. Substrate concentration versus initial velocity (Vo) and non-linear regression plots for the determination of glycine and succinyl-CoA Kms. Glycine concentration vs initial velocity for rHemT with the corresponding non-linear regression plot (R2 value is 0.98) (A), and succinyl-CoA concentration vs initial velocity for rHemT with the corresponding non-linear regression plot (R2 value is 0.99) (B).

Table 2. Kinetic parameters determined for rHemT. Kinetic parameters rHemT

Vmax for glycine (mol ALA/h) 1.15

Km for glycine (mM) 9  2

-1 kcat for glycine (h ) 50

-1 kcat/Km for glycine (mM h ) 6

Vmax for succinyl-CoA (mol ALA/h) 2.7

Km for succinyl-CoA (M) 18  3

-1 kcat for succinyl-CoA (h ) 68

-1 kcat/Km for succinyl-CoA (mol h ) 4

Is HemT an ALA synthase or a KBL? Recently, it has been shown that, although hemT is transcriptionally silent in R. sphaeroides wild type strain 2.4.1, the gene is actively transcribed in R. sphaeroides strain 2.4.9, and expression is maximal in cells grown under anaerobic dark conditions with dimethyl sulfoxide provided as alternate electron acceptor (12). 26

However, the preliminary data suggest that, when the hemT gene is disabled, growth is

unimpaired (10). Therefore, the contribution of HemT to ALA metabolism is not yet resolved.

The enzyme 2-amino-3-ketobutyrate CoA ligase, or KBL, is a close structural relative of

ALA synthase; both are members of the aspartate aminotransferase fold type I superfamily of

PLP-dependent enzymes. The forward reaction catalyzed by this enzyme is the second in the

threonine utilization pathway, which is an alternate pathway for serine biosynthesis (28, 36).

The predicted similarities in structure are suggested by superimposing models of rHemT and R.

sphaeroides KBL (234/396 amino acid sequence identities with E. coli MG1655 KBL) protein,

shown in Fig. 10. Expression of the kbl gene has not been evaluated in any strain of R. sphaeroides, nor has the contribution of the threonine utilization pathway to serine production.

At the amino acid sequence level R. sphaeroides KBL is more similar to HemT (127/357 identities) than HemA (115/331 identities). Further, as will be discussed in more detail later

(Chapter 3), while the rHemA and rHemT Kms for glycine are the same (50), the rHemA Km for succinyl-CoA is half that of rHemT protein (50). These considerations, as well as the absence of a phenotype for hemT null mutants (L. Cooper and J. Zeilstra-Ryalls, unpublished observations, ref.10), and a report of substrate promiscuity for a member of the same enzyme superfamily present in Thermus thermophilus (19), suggested that an evaluation of HemT substrate preference, especially whether or not the enzyme can act upon acetyl-CoA (KBL activity) was warranted. As reported in Table 3, no KBL activity was detected for purified rHemT.

Therefore, the data indicate that HemT is a far better ALA synthase than a KBL. 27

Figure 10. Tertiary structure superposition of R. sphaeroides 2.4.1 rHemT and KBL. The model of rHemT is in red, and that of KBL (RSP_2376) is in yellow. The figure was generated using LOMETS (49) and Tm-align (56).

Table 3. Specific enzyme activity of rHemT using glycine with succinyl-CoA versus glycine with acetyl-CoA. Substrate Specific activity a/mg) (U

succinyl-CoA 25 ± 3

acetyl-CoA 0 ± 3

aU is Units, defined as described in the footnote to Table 1.

Circular dichroism (CD) analysis of rHemT secondary structure. Towards confirming the similarities in structure suggested by the models of rHemA and rHemT, the secondary structure of the purified protein was examined by CD. This was also important for assessing whether the polyhistidine tag has any significant effect on the secondary structure of rHemT which may affect the enzyme activity. The CD spectrum (Fig. 11) had a profile resembling that of proteins having significant amounts of alpha-helix structural elements. The 28

percentage alpha helical content calculated using equations 4 and 5 (Materials and Methods, ref.

11), and by using the K2D algorithm (1) are reported in Table 4. The percent content of beta sheet and random coil, according to the K2D program are also included, as well as predicted values for both wild type and rHemT protein using LOMETS (49). The predicted and calculated values for rHemT are similar to each other and to the predicted values for wild type HemT protein. They are also similar to the values for the solved structure of Rhodobacter capsulatus

ALA synthase, which is 44% alpha helix, 16% beta sheet and 40% random coil.

Table 4. Theoretical and calculated secondary structure values for rHemT.

Method -helix (%) -sheet (%) random coil (%)

208 nm 36±0.6 NDa ND

222 nm 34±0.9 ND ND

K2D 35±0.7 20±0 45±1.4

LOMETS 42 13 45

aND: not determined; these absorbances are specific for -helical structures.

Figure 11. Circular dichroism spectra of rHemT in the far UV region (200–250nm). The CD spectra using 0.34 mg/ml protein in 20 mM tricine, 10% glycerol, 25 mM NaCl, pH 7.2, fitted to a running average (A) and fitted using the K2D program (1) (B). In both cases, raw data are dots while the solid curve is the fitted data.

Disulfide bond determination. Previous studies have shown that ALA synthase activity

is responsive to treatments that reduce disulfides; i. e. activity is increased by reducing agents

(9). Further, when HemA is exposed to alkylating agents under reducing conditions, activity is

reduced up to 70% (6). Since this loss of activity does not occur in the presence of succinyl-CoA

(6), it is thought that at least one cysteine residue within the succinyl-CoA binding site is important for function. A study of the presence of disulfides within rHemT was undertaken for two reasons. Initially, there was concern that, upon storage, protein oxidation might contribute to the rapid loss of activity that was observed for dilute purified protein. Stability improved for concentrated protein, and so oxidation was probably not relevant. However, that wild type

HemT has five cysteines, only three of which are at positions corresponding to three of the five cysteines also present in HemA. This suggested that it would be important to examine how many HemT cysteines, if any, participate in disulfide bond formation, in order to compare this feature between the HemT and HemA proteins. 30

To completely reduce all the cysteine residues, rHemT was incubated with 50 mM

dithiothreitol for 10 min, and to completely oxidize the protein it was incubated with 10 mM

hydrogen peroxide for 10 min. The presence of disulfide bonds was then compared for treated

and untreated protein using two methods. One involves treatment with 4-acetamido-4’- maleimidylstilbene-2,2’-disulfonic acid (AMS) and the second treatment with 4-chloro-7- nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), as described in Materials and Methods. Detection of

AMS-modified protein is by SDS-PAGE, as the molecular weight of the protein increases by approximately 500 daltons for each residue that is modified. NBD-Cl modification is detected spectrophotometrically, as upon reaction with a thiol, the thioether gives a characteristic

absorbance at 424 nm (5).

As shown in Fig. 12, while the mobility of the protein decreased after modification with

AMS (compare lanes 2 to 3 and 4), there is no apparent difference following oxidation (lane 3)

versus reduction (lane 4). This indicates rHemT does not contain disulfides. Optimization of the

protocol for NBD-Cl, which will be used to confirm the AMS modification results, is in

progress, and no results are as yet available.

Figure 12. SDS-PAGE gel of rHemT treated with AMS. Lane 1: molecular mass marker, lane 2: rHemT, lane 3: oxidized rHemT treated with AMS, lane 4: reduced rHemT treated with AMS.

DISCUSSION

Previous efforts to purify HemT in its active form have not been successful. It is presumed that this was due to the insolubility of the purified recombinant enzyme (6).

Therefore, HemT has remained completely uncharacterized to date. The expression system used here, involving a native as opposed to heterologous bacterial host, resulted in production of soluble rHemT that has been successfully purified in active form. Approximately 37 mg of pure rHemT protein with a specific activity of 26 ± 4 U/mg (where 1 unit is defined as 1 µmol ALA formed per hour at 37oC, pH 7.2) was obtained from 1 liter of culture (OD 0.8-1.0).

Based on CD analysis, the purified protein is similar in structure to the solved crystal

structure of R. capsulatus ALA synthase. This suggests that the presence of the additional amino

acid residues comprising the affinity tag used for purification does not grossly affect the protein structure.

Preliminary evidence also indicates that rHemT lacks cystines, and so the previous observations regarding enzyme activation by reducing agents has not been substantiated.

However, further confirmation is required.

Although its preference for other substrates should be fully explored, the present study has shown that purified rHemT is a better ALA synthase than a KBL. This suggests that the question remains as to what contribution HemT makes to ALA biosynthesis. This question may be addressed by comparing the properties of this enzyme to that of HemA, the other R. sphaeroides ALA synthase. 32

CHAPTER 3: COMPARISON OF R. sphaeroides 2.4.1 POLYHISTIDINE-TAGGED HemA WITH POLYHISTIDINE-TAGGED HemT

INTRODUCTION

In parallel with the purification and characterization of recombinant polyhistidine-tagged

HemT (rHemT) described in the previous chapter of this thesis, recombinant polyhistidine- tagged HemA (rHemA) protein was purified and characterized in the lab (X. Xiao, MS Thesis, submitted, ref. 50). In this chapter the characteristics of the two proteins will be compared and contrasted for the purpose of developing hypotheses as to the significance of the presence of these ALA synthase isoenzymes in the cell.

PROPERTIES OF PURIFIED R. sphaeroides 2.4.1 rHemA versus rHemT

The amino acid sequences of both HemA and HemT proteins are 407 amino acid residues in length. The rHemT polypeptide is 424 amino acids long, while rHemA is 433 amino acid long. It is clear from an alignment of the amino acid sequences of HemA and HemT, together with the R. capsulatus ALA synthase sequence, shown in Fig. 13, that the proteins are highly conserved. Further, amino acid residues that are reported to be important for the enzymatic activity of R. capsulatus ALA synthase are present in both HemA and HemT. These include lysine 248 (highlighted in blue in Fig. 13), which binds to the aldehyde group of PLP to form the

Schiff-base intermediate, and so is central to the catalytic activity of the enzyme, and arginine

374 (highlighted in green in Fig. 13), involved in binding the carboxyl group of glycine (16, 55).

The region that is responsible for binding succinyl-CoA, GAGSGGTRNISGT (highlighted in gray in Fig. 13) is also highly conserved. Therefore, those residues known to be critical for ALA synthase function are present in both HemA and HemT.

HemT MEFSQHFQKL IDDMRLDGRY RTFAELERIA GEFPTALWHG PDGQARRVTV WCSNDYLGMG HemA MDYNLALDTA LNRLHTEGRY RTFIDIERRK GAFPKAMWRK PDGSEKEITV WCGNDYLGMG Rcaps MDYNLALDKA IQKLHDEGRY RTFIDIEREK GAFPKAQWNR PDGGKQDITV WCGNDYLGMG *:::::::+: + :: :*** ***:::** : *:**:* * *** :** **:*******

HemT QNAEVLAAMH RSIDLSGAGT GGTRNISGTN RQHVALEAEL ADLHGKESAL IFTSGWISNL HemA QHPVVLGAMH EALDSTGAGS GGTRNISGTT LYHKRLEAEL ADLHGKEAAL VFSSAYIAND Rcaps QHPVVLAAMH EALEAVGAGS GGTRNISGTT AYHRRLEAEI ADLHGKEAAL VFSSAYIAND *:::**+*** :::^ ***: *********: :* :****+ *******:** :*:*::*:*:

HemT AALGTLGKIL PECAIFSDAL NHNSMIEGIR RSGAERFIFH HNDPVHLDRL LSSVDPARPK HemA ATLSTLPQLI PGLVIVSDKL NHASMIEGIR RSGTEKHIFK HNDLDDLRRI LTSIGKDRPI Rcaps ATLSTLRVLF PGLIIYSDSL NHASMIEGIK RNAGPKRIFR HNDVAHLREL IAADDPAAPK *:*:** : *:: * ** * **:******^ *^^ ^: ** *** +*:^+ ^ ^ +++^*+

HemT IVAFESVYSM DGDIAPIAEI CDVAERHGAL TYLDEVHAVG LYGPRGGGIS DRDGLADRVT HemA LVAFESVYSM DGDFGRIEEI CDIADEFGAL KYIDEVHAVG MYGPRGGGVA ERDGLMDRID Rcaps LIAFESVYSM DGDFGPIKEI CDIADEFGAL TYIDEVHAVG MYGPRGAGVA ERDGLMHRID :^******** ***::+* ** **:*:::*** +********* :*****^*:: :****:+*::

HemT IIEGTLAKAF GVMGGYVSGP SLLMDVIRSM SDSFIFTTSI CPHLAAGALA AVRHVKAHP. HemA IINGTLGKAY GVFGGYIAAS SKMCDAVRSY APGFIFSTSL PPVVAAGAAA SVRHLKGD.. Rcaps IFNGTLAKAY GVFGGYIAAS AKMVDAVRSY APGFIFSTSL PPAIAAGAQA SIAFLKTAEG *^:***+**: **:***:::: ^:: *::**: :::***:**: :* **** * :^^^:*

HemT .DERRRQAEN AVRLKVLLQK AGLPVLDTPS HILPVMVGEA HLCRSISEAL LARHAIYVQP HemA VELREKHQTQ ARILKMRLKG LGLPIIDHGS HIVPVHVGDP VHCKMISDML LEHFGIYVQP Rcaps QKLRDAQQMH AKVLKMRLKA LGMPIIDHGS HIVPVVIGDP VHTKAVSDML LSDYGVYVQP :* +: * **::*: :*+*::*::* **:** ^*:: ::^: ^*::* * :^****

HemT LARHAIYVQP INYPTVARGQ ERFRLTPTPF HTTSHMEALV EALLAVGRDL GWAMSRRAA HemA LEHFGIYVQP INFPTVPRGT ERLRFTPSPV HDSGMIDHLV KAMDVLWQHC ALNRAEVVA Rcaps LSDYGVYVQP INFPTVPRGT ERLRFTPSPV HDLKQIDGLV HAMDLLWARC ALNRAEASA * :^**** **:***:**: **:*:**:*: *: :: ** *:: :: : :::::: *

Figure 13. Amino acid sequence alignment of wild type HemT and HemA, together with the R. capsulatus ALA synthase (Rcaps) The sequences were aligned using ClustalW with default settings (Blosum scoring matrix, opening and ending gap penalty values of 10, extending gap and separation gap penalty values of 0.05). The catalytic lysine residue, conserved in all ALA synthases (18), is highlighted in blue, the arginine involved in glycine binding is highlighted in green, and the sequences involved in binding of succinyl-CoA are shaded grey. Symbols are: *, identities between all three proteins; identities between HemA and R. capsulatus ALA synthase; +, identities between HemT and R. capsulatus ALA synthase; ^, identities between HemA and HemT.

The amino acid sequence conservation between HemA and HemT predicts structural

similarity, and based upon modeling (Fig. 14), the secondary structures of the proteins are very similar to each other, and also to R. capsulatus ALA synthase (2). In addition, the preliminary circular dichroic measurement of -helical, -sheet, and random coil content of rHemA protein are 40%, 12%, and 48% versus 35%, 20%, and 45% for rHemT. Certainly the amino acid residues that are the result of engineering of the expression constructs contributes to these 34

structural differences, and so it is likely that the wild type proteins bear an even greater structural resemblance to each other.

Figure 14. Tertiary structure model superpositions of R. sphaeroides 2.4.1 ALA synthases and R. capsulatus ALA synthase (16). In (A) the model of R. sphaeroides wild type HemT (yellow) is superimposed upon the solved crystal structure coordinates of R. capsulatus ALA synthase (red). In (B), the model of R. sphaeroides wild type HemT (red) is superimposed upon the model of R. sphaeroides wild type HemA (yellow). The models were generated using LOMETS (49) and the superimposed images were generated using TM-align (56).

The two purified enzymes have the same optimum pH (7.2) and temperature (37oC).

Also, the effect of hemin on ALA synthase activities of the two proteins is similar, although rHemT is somewhat more sensitive than rHemA (the addition of 200 M hemin reduces rHemT activity by approximately 35%, while rHemA activity is reduced by approximately 30% by 100

M hemin addition).

It is from a comparison of the kinetic properties of the two proteins that differences between rHemT and rHemA become apparent (Table 5 and ref. 50). In terms of specific activity, turnover number (kcat), and its catalytic efficiency (kcat/Km) rHemA is a far better ALA synthase

than rHemT. Also, while the Km for glycine for the two enzymes is almost the same, the rHemA 35

Km for succinyl-CoA is 2-fold lower than that of rHemT. Given its relatively poor behavior as

an ALA synthase, it is possible that HemT may actually fulfill a different enzymatic role in the

cell, and so its preferred substrates may be compounds other than glycine and succinyl-CoA.

Since the rHemT affinity for glycine is the same as that of rHemA, the more likely alternative

substrate is a different CoA derivative. In this study, the ability of rHemT to function as a KBL,

which combines glycine with acetyl-CoA, was assessed. No product was detected, and so HemT

is probably not a KBL enzyme. Whether or not rHemT prefers other CoA derivatives remains to

be determined. They were not considered here since they are not commercially available.

However, in vivo methods can be used to assess this possibility and are underway.

Table 5. Comparison of the kinetic properties of Rhodobacter sphaeroides 2.4.1 rHemA and rHemT. Property rHemT rHemAa Specific activity (µmol ALA/h/mg) 30±4 624±45 Succinyl-CoA Km (M) 18.0±3 7.8±1.7 Succinyl-CoA Vmax (µmol ALA/h) 2.7 1.0 -1 Succinyl-CoA kcat (h ) 67.5 635.2 -1 Succinyl-CoA kcat/Km (M h ) 3.8 80.0 Glycine Km (mM) 9.0±2.0 8.7±1.3 Glycine Vmax (µmol ALA/h) 1.15 0.80 -1 Glycine kcat (h ) 50.0 1377.7 -1 Glycine kcat/Km (mM h ) 6 153 aValues are from ref. 26.

Previous studies have shown that reduced cysteines are involved in the activation of ALA

synthase activity in R. sphaeroides (9). To investigate whether, during purification the rHemT

acquired a cystine that has reduced its activity, the number of disulfide bonds following

oxidizing and reducing treatments was examined. Preliminary results indicate that following the

two treatments there is no difference in the number of free cysteines available for modification 36

by AMS, and so indicates that the low activity of the protein is not due to an oxidation-dependent event.

DISCUSSION

A comparison of the properties of the polyhistidine-tagged proteins indicates that HemT is a low-activity ALA synthase. What purpose would an apparently poorer enzyme have in the cell when another high activity enzyme is also present? A working hypothesis has been formulated based on a consideration of the differential expression of the hemA and hemT genes

(12), and the fact that the substrates for ALA synthases, glycine and succinyl-CoA, participate in a multitude of metabolic pathways. When cells are transitioning from one energy "lifestyle" to another, it is important that the use of metabolites that participate in both catabolic and anabolic metabolisms be appropriately balanced. As these cells transition from aerobic to anaerobic respiration with dimethyl sulfoxide as alternate electron acceptor considerable metabolic remodeling is required, which relies upon protein synthesis and so demands glycine availability and energy which would need to come from substrate-level phosphorylation. During this same transition, both hemA and hemT transcription is upregulated. It is necessary to point out that this only happens in strain 2.4.9, as hemT is not transcribed in strain 2.4.1 under any condition tested.

While it has been well-established that R. sphaeroides strain 2.4.1 responds to the absence of oxygen even in the dark by developing photosynthetic membranes towards the possible presence of light as an energy source (and so a dramatic increase in ALA production makes sense because it is required to produce the large amounts of tetrapyrroles, primarily bacteriochlorophyll, required for the photosynthetic apparatus), there is little knowledge as to how strain 2.4.9 responds to oxygen availability versus light availability. That it may not be the same as for strain 37

2.4.1 is suggested by studies in the lab of J. Zeilstra-Ryalls that have demonstrated that hemT

transcription is only upregulated under anaerobic-dark conditions, while hemA transcription is

induced regardless of the presence or absence of light.

The hypothesis is that, when both HemA and HemT polypeptides are being made in

maximum amounts – anaerobic dark conditions – HemT polypeptide can form a dimer with

HemA polypeptide. In this way the overall ALA synthase activity is lowered relative to that when HemT is reduced in concentration in the cytoplasm – anaerobic light conditions. This novel regulatory mechanism remains to be demonstrated. But having the ability to produce active HemT enzyme makes such a demonstration feasible. Thus, one could construct an expression system capable of producing both polyhistidine-tagged HemT and a recombinant

HemA protein having a different tag. It should be possible to capture the heterodimers, if present, by affinity chromatography involving one or the other tag. They could then be

characterized in the same manner as has been described here for rHemT and elsewhere (X. Xiao,

MS Thesis, submitted, ref. 50) for rHemA. Evidence that the heterodimers have catalytic

properties intermediate to those of each homodimeric protein would support the hypothesis, and

suggest that HemT functions to negatively modulate ALA production, and so redirects succinyl-

CoA and glycine to other metabolisms. 38

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