Characterization, Mutagenesis and Mechanistic Analysis of the Sterol C24- Methyltransferase: Implications for Understanding Active Site Requirements for Sterol Biosynthesis
by
Alicia L. Howard, M.S.
A Dissertation
In
Biochemistry
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirement for the degree of
Doctor of Philosophy
Approved
Dr. W. David Nes Chair of the Committee
Dr. Joachim Weber
Dr. Paul W. Pare
Dr. Mark Webb Dean’s Representative
Mark Sheridan Dean of the Graduate School
December, 2016 Copyright 2016, Alicia L. Howard Texas Tech University, Alicia L. Howard, December 2016
ACKNOWLEDGMENTS
First, I would like to extend my deepest gratitude to my mentor, Paul Whitfield Horn Professor, Dr. W. David Nes for all of his patience, guidance, support, and encouraging words when it was needed most. I would also like to thank Dr. Joachim Weber and Dr. Paul W. Pare, Associate professors of Chemistry and Biochemistry at Texas Tech University, for sitting on my committee to provide guidance and helpful comments on my dissertation. I thank Dr. Mark Webb for being my Deans Representative for my doctoral defense. I would like to express my deepest gratitude to the faculty and staff of the Department of Chemistry and Biochemistry at Texas Tech University for assisting me in my research. I would also like to express my sincere appreciation to the following individuals who have been a part of the Nes lab during my time at Texas Tech University: Mr. Boden Vanderloop, Mr. Matthew Miller, Mr. Presheet Patkar, Ms. Maja Milankovic, Ms. Madison Keller, Mr. Matthew Sowa, Mr. Garrett Morh, and Mr. Brad Haubrich, for always supporting and assisting me throughout my research. I would also like to thank The National Institutes of Health and Texas Tech Graduate School for both supporting me with a fellowship, allowing me to focus on my research. I would like to give the biggest thanks to my beloved friends who have been there to encourage and push me through hard times when I desperately needed it: Whitney Pierce, Crista Thomas, Christina Phan, Stephanie Ramos, Jimmy Jackson, Taylor Ridge, Alauna Hunter, and Eric Davila. Life would be a lot less colorful without all of you in it. I would like to send all my love to my amazing, strong, inspiring parents, Sherman and Alicia A. Howard; and my two older brothers and sister, Rodney, Sherman, and Janice for all of the love, laughs, and tears that have helped sustain me in this thing called life. Lastly, but far from least:
God Almighty is worthy of all acknowledgements……………….
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TABLE OF CONTENTS
ACKNOWLEDGMENTS ii
ABSTRACT v
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS xii
CHAPTERS
I. INTRODUCTION 1
1.1 Sterol Evolution 1
1.2 Sterol Biosynthesis 7
1.3 Functional Domain of Sterols 10
1.4 Sterol Methyltransferase 14
1.5 Substrate Specificity and Analogs 19
1.6 Research Objections 22
II. EXPERIMENTAL METHODS 24
2.1 Chemicals and Solvents 24
2.2 Substrates 24
2.3 Yeast Culture and Growth Conditions 24
2.4 Analysis of Sterol at IC50 25
2.5 Functional Expression of Cloned C24-SMT 26
2.6 Standard Enzyme Assays 27
2.7 Protein Quantification: Bradford Method 28
2.8 Preparation Plasmid 28 iii Texas Tech University, Alicia L. Howard, December 2016
2.9 Site-directed Mutagenesis 29
III. Saccharomyces cerevisiae Growth Studies 31
3.1 Previous Research with Fungal C24-SMT 31
3.2 Sterol Composition 34
3.3 Inhibitors 38
3.4 25-Azalanosterol 42
3.5 Abasol 46
IV. Mutagenesis of the Sterol C24-Methyltransferase 50
4.1 ATP Binding Region 50
4.2 SMT Sequence Comparison and Mutagenesis 57
4.3 The Significance of Tyr81 Mutants 63
4.4 Acidic and Histidine Residue Mutations 66
4.5 Homology Model 67
4.6 Molecular Docking 71
V. DISCUSSION 78
REFERENCES 81
APPENDICES 85 A. MASS SPECTRA OF STUDY SUBSTRATES 85
B. OLIGONUCLEOTIDES SEQUENCES 89
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ABSTRACT
Previous studies that have defined regions of the sterol C24-methyltransferase (SMT) primary structure involved with catalysis failed to show the full identity of essential amino acids associated with the sterol binding segments that contribute to C- methylation from SMTs. Here we report the characterization and inhibition of the C24- SMT from Saccharomyces cerevisiae and Chlamydomonas reinhardtii, a crucial enzyme responsible for the C1 transfer in the 24-alkyl side chain construction of phytosterols. A series of 27 mutants across kingdoms were evaluated for product outcome to locate relevant residues and regions of the SMTs. These mutations converted substrate to product ratios that favored the formation of Δ25(27)-olefins over the preferred Δ24(28)- olefins found in the wild type SMT and revealed a few essential amino acids confirm by a loss of activity. Two transition state-based and product inhibitors evaluated with ScSMT
were shown to inhibit SMT activity with IC50 values ranging from 1 µM (25-
azalanosterol, a transition state analog) to 7 μM (abasol, a product inhibitor). These IC50 values are similar to antifungals established for opportunistic pathogens. By using inhibitors, sequence alignments of the SMT and site-directed mutagenesis of select residues within the conserved regions of the primary structure, a homology model of Trypanosoma brucei SMT was made that incorporates all of the key structural features of the enzyme in its mechanism of binding to the sterol based on previous binding studies with T. brucei and S. cerevisiae. From the combination of results, the identity of amino acid residues in the active site and inhibition profiles provide a closer look into the catalysis of the sterol C24-SMT.
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LIST OF TABLES
1. Catalytic competence of sterols to the PbSMT. 21
2. Sterol composition of Saccharomyces cerevisiae. 42
3. Antimicrobal activity of 25-azalanosterol against S. cerevisiae. 44
4. Antimicrobal activity of abasol against S. cerevisiae. 48
5. The Walker sequence across kingdoms. 52
6. Catalytic competence of S. cerevisiae mutants. 54
7. Catalytic competence of C. reinhardtii and G. max mutants. 57
8. Catalytic competence of C. reinhardtii Tyr110 mutants. 66
9. Catalytic competence of S. cerevisiae mutants made in the Nes lab. 75
10. Catalytic competence of fungal mutants made in the Nes lab. 76
11. Catalytic competence of plant mutants made in the Nes lab. 77
12. DNA sequence of the synthetic oligonucleotides used for site-directed mutagenesis of the ERG6 cDNA 89
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LIST OF FIGURES
1. Presqualene cholesterol synthetic pathway. Lanosterol is synthesized from acetate in a series of enzymatic reactions. 2
2. Generalized structure and numbering of the sterol molecule (adapted from Nes and Venkatramesh, 1994). 3
3. Sterols characterized from organisms other than fungi, vertebrates, and land 5
4. Evolutionary split between prokaryotes and eukaryotes in the biological pathway for their respective membrane sterols. 6
5. Generalized synthetic pathway of sterols. Sterol precursor squalene is oxidatively converted to oxidosqualene, which is cyclased to one of two protosterols: cycloartenol or lanosterol. The protosterol undergoes subsequent modifications including oxidative demethylations and desaturations to result in the terminal sterol product. Enzymes are labelled with EC number where available, or gene abbreviation. Terminal sterols yield derived steranes after burial and diagenesis. Enzymes labelled in bold are discussed in the text. Those requiring molecular oxygen are noted. 8
6. The representative phytosterols (24-alkyl sterols) with the different side chains, panel A, that ultimately lead to ergosterol and sitosterol (5). In panel B, the preferred substrates for the C24-SMT. 9 7. Diagram of the four regions of importance for membrane insertion as illustrated on ergosterol (fungal sterol). Domain 1 is the equatorial 3- hydroxyl group; Domain 2 is the planar tetracyclic ring system also known as the nucleus; Domain 3 is the right-handed C20R configuration; Domain 4 is the C8- or C10-side chain. 11 8. Conformational perspective of relevant sterols illustrating the flat structure and the tilt of the C3 and C17. 12 9. Diversity of 24-alkyl sterol biosynthesis scheme. The final sterols brassicasterol and stigmasterol can be formed by different routes that occur in protozoa, fungi and plants. 13 10. SMT1 and SMT2 are distinguished by substrate specificity toward the Δ²⁴⁽²⁵⁾- and Δ²⁴⁽²⁸⁾-olefin side chain structure. The gene families are
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grouped into five distinct subfamilies based on sequence relatedness and substrate preference, as indicated in the figure. 15
11. Ergosterol biosynthetic pathway in fungi showing all the necessary steps for the production of a membrane inserted sterol 16
12. The steric-electric plug model of the sterol methyltransferase binding and catalysis of sterol and AdoMet; SMT: sterol methyltransferase enzyme, E: enzyme and S: substrate. 18
13. The parent structure (1), lanosterol, and the compounds assayed the with varied C3 residues labeled in table 1. 19
14. Variations in sterol profiles of C. glabrata isolates 21231 (wild type) and 21229 (mutant). Sterols of the heptanic fraction were analyzed by gas chromatography. As highlighted by the dashed line, ergosterol, which was the major sterol species for isolate 21231 (A), was not detectable on the chromatogram of isolate 21229 (B). (C) Percentages of the ergosterol biosynthesis intermediates determined from the corresponding peak areas and retention times. 33
15. Alignment of sterol C24-methyltransferase amino acid sequence sequences (GenBank accession numbers) from C. glabrata (AAX73200.1) and S. cerevisiae (NP_013706.1). Identical residues conserved in the primary structure are shaded red. The position of the C198F mutant is indicated by the arrow. 36
16. The growth curve of S. cerevisiae over a course of 48hrs (panel 1). The GC-MS analysis of the product distribution of ScSMT wild type (panel 2). The letters over each peak in panel 2 corresponds with the sterols in the table (panel 3). 37
17. The biosynthesis of ergosterol and cholesterol showing the main steps, the enzymes involved, and the known inhibitors. 39
18. The structures of the inhibitors tested alongside the native substrate they mimic. 41
19. Inhibitors tested with the SMT: A. Native C-methylation reaction progress; B. Rationally designed inhibitors. 41
20. The sterol compositions in panel 2 at the IC50 of 25-azalanoster (panel 1) with peaks A) zymosterol, B) Δ5,7,24-cholestatrienol, C) Δ7-
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zymosterol and D) Δ5,7,22,24-cholestatetraenol. Sterol fractions were analyzed by gas chromatography. 44
21. Comparison of the sterol profile of yeast wild type (panel 1-A) against yeast incubated with abasol (panel 1-B) with that of C. glabrata isolates 21231 (2-A, wild type) and 21229 (2-B, mutant). Sterol fractions were analyzed by gas chromatography. Both show erogsterol (A) as the major peak, which was no longer detectable with an increase of zymosterol in the mutant and the yeast incubated with abasol (B). Peak B and D in panel 1-B appears to correspond to peak 4 and 6 in panel 2- B as cholesta-5,7,24-trienol and cholesta-5,7,22,24-tetraenol, respectively, as the Δ5,7-dienols detected in the 21229 isolate. 45
22. The sterol compositions in panel 2 at the IC50 of 25-azalanoster (panel 1) with peaks A) zymosterol, B) Δ5,7,24-cholestatrienol, C) Δ7- zymosterol and D) Δ5,7,22,24-cholestatetraenol. Sterol fractions were analyzed by gas chromatography. 49
23. The primary sequence of ScSMT, accession number gi|6323635 50
24. GC-MS of the ScSMT wild type (control) conversion of cycloartenol to 24(28)-methylene cycloartenol. In panel 2 is the mass spectra of the product peak labeled B eluding in panel 1. The substrate peak is labeled A. 53
25. GC-MS of the ScSMT G347N conversion of cycloartenol to 24(28)- methylene cycloartenol. G347N represents the category: 40% or greater resulting in sigmoidal behavior with an ATP affect. In panel 2 is the mass spectra of the product peak labeled B eluding in panel 1. The substrate peak is labeled A 53
26. GC-MS of the ScSMT G347N conversion of cycloartenol to 24(28)- methylene cycloartenol. G347N represents the category: 20-30% or greater resulting in sigmoidal behavior with no ATP affect. In panel 2 is the mass spectra of the product peak labeled B eluding in panel 1. The substrate peak is labeled A. 54
27. GC-MS of the CrSMT wild type (control) conversion of cycloartenol to cyclolaudenol and 24(28)-methylene cycloartenol. In panel 2 is the mass spectra of the product peaks labeled B and C eluding in panel 1. The substrate peak is labeled A 56
28. GC-MS of the GmSMT1 wild type (control) conversion of cycloartenol to 24(28)-methylene cycloartenol and triplet of 24-ethyl olefins. In ix Texas Tech University, Alicia L. Howard, December 2016
panel 2 is the mass spectra of the product peaks labeled B and C eluding in panel 1. The substrate peak is labeled A. 56
29. The C24-SMT sequence alignment across kingdoms (GenBank accession numbers) from S. cerevisiae (gi|6323635), C. reinhardtii (gi|15965129), G.max SMT1 (gi|351725990), G. max SMT2 (gi|242755468) and T. brucei (gi|70832598). The five conserved regions are boxed in green. 59
30. Proposed aspects of substrate (A) intermediate (B) and product (C) binding to the PbSMT active site. For lanosterol binding in A, the enzyme presents a binding site that is sterically and electronically complementary, to which the substrate becomes anchored at its C3- hydrophilic group. The sterol C3–OH group interacts in a pre-organized active site with contacts that form hydrogen bonds against the 3-oxygen atom (from a main frame moiety, M) and hydrogen atom of the 3- oxygen atom (from a basic amino acid, B1) forming a hydrogen bonded network to stabilize the ground state structure at the proximal end of the acceptor molecule and the side chain assumes a pseudocyclic conformation. Productive orientation of the substrate side chain affords backside (SN2) addition of “methyl cation” from S-adenosyl-l- methionine (represented by the catalytic sulfur atom, S) to the Δ24- bond generating the C24β-methyl C25 cation shown in B. Deprotonation of the C28 methyl group from a basic amino acid (B2) can lead to the C24(28)-methylene product shown in C, followed by disassociation of the methylated sterol from the enzyme. 61
31. The general mechanism of site-directed mutagenesis with the mutated codon highlighted in pink and the parent codon in blue. 62
32. The predicted secondary structure of SMT showing α-helices, β-sheets and loops. The relevant secondary structural units are numbered by their position in the alignment shown in Figure 29. The four conserved substrate binding domains are labeled I to IV with sterol acceptor and methyl donor docked in the model based on the results of several lines of evidence discussed in the text. A hydropathy plot for the S. cerevisiae SMT is shown illustrating the location of the substrate binding segments indicated in the model above. 68
33. Ribbon representation of the active site of TbSMT. The AdoMet- (SAM; magenta) and sterol- (STE; orange) binding pockets are shown. Tyrosine residues that were substituted are labelled in blue. Relevant contact amino acids identified in the homology modelling are shown in stick representation. The image was using PyMol with coordinates
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obtained by homology modelling against CMA coordinates as described in Materials and methods section. 69
34. Alignment of sterol C24-methyltransferase amino acid sequence sequences (GenBank accession numbers) from L. infantum (ADX32472.1) and T. brucei (AAZ40214.1). Conserved regions are boxed in green with residues highlighted in red corresponding to Azams model and the blue highlighted residues corresponding to the TbSMT model. 72
35. Interactions of 24(R,S),25-epiminolanosterol in the binding site of SMT enzyme are shown in 2D conformation with surrounding residues using TbSMT amino acid numbering. 73
36. Mass Spectrometer for the sterol composition found in ScSMT wild type growth studies; panel A) Zymostero; panel B) Ergosterol; panel C) Fecosterol panel D) Δ7-Fecosterol 85
37. Mass Spectrometer for the sterol composition found in ScSMT wild type growth studies when incubated with inhibitors; panel A) Zymostero; panel B) Δ5,7,24-Cholestatrienol; panel C) Δ7-Zymosterol; panel D) Δ5,7,22,24-Cholestatetraenol 87
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LIST OF ABBREVIATIONS
°C: degrees celsius
CrSMT: Chlamydomonas reinhardtii sterol methyltransferase ddH2O: double distilled water
E. coli: Escherichia coli
GC: gas chromatography
HPLC: high performance liquid chromatograpgy
IC50: inhibitor concentration at 50% inhibition
IPTG: Isopropylβ-D-thiogalactopyranoside kDa: kilodalton
KOH: potassium hydroxide mL: mililiters
MS: mass spectrometer
M/Z: mass to charge nM: nanomolar
PbSMT: Paracoccidioides brasiliensis sterol methyltransferase
SAM: S-adenosyl-L-methionine
ScSMT: Saccharomyces cerevisiae sterol methyltransferase
SMT: sterol C24-methyltransferase
TbSMT: Trypanosoma brucei sterol methyltransferase
μM: micromolar
Yeast SMT: Saccharomyces cerevisiae sterol methyltransferase
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CHAPTER I INTRODUCTION
1.1 Sterol Evolution Triterpenoids are a large family of lipids that are widely distributed in bacteria (hopanoids) and eukaryotes (sterols). Prokaryotes form the pentacyclic hopanoid for their cell membrane insert by coiling left-handedly to form (+)-tetrahymanol anaerobically, the absence of oxygen (1). The product can then be directly introduced to the prokaryotic cell membrane. Sterols are a class of triterpenoids derived from the C₃₀ squalene that are generated from the linear combinations of the C₅-isoprenoid building block, isopentyl diphosphate, (Figure 1) (2). Sterols possessing the cyclopentanophenathrene ring form the sterolome that comprises a chemical library of more than 1000 natural products found in all forms of eukaryotes and some prokaryotes that provide a number of biological functions (Figure 2) (2). They are present in all eukaryotes where they are essentially involved in both intra- and intercellular signaling and in the organization of membranes where they affect fluidity and permeability (3). Cell wall biosynthesis and maintenance is a highly dynamic process that is tightly regulated during cell growth and morphogenesis, and it responds to a variety of cell stresses (4). It involves modification of existing biosynthetic machinery through interactions with cell stress and cell integrity pathways, as well as delivery of new enzymes from the golgi complex through the secretory vesicle system (4). Cholesterol is the only sterol that is inserted across the phospholipid bilayer in the human body, which differs from the membrane inserts ergosterol in fungi and sitosterol in plants (5).
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Figure 1. Presqualene cholesterol synthetic pathway. Lanosterol is synthesized from acetate in a series of enzymatic reactions (6).
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Figure 2. Generalized structure and numbering of the sterol molecule (adapted from Nes and Venkatramesh, 1994) (7).
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The diversity and nature of sterols and the pathways leading to these compounds have been thoroughly studied in vertebrates, fungi, and lands plants (3). Characterizations of sterols from other organisms have unveiled a wide variety of molecules, among which not only the same sterols known in animals, fungi, and land plants but also other types of sterols with insaturations at various positions of the cycle or in the side chain, and possibly alkylations or inclusion of a cyclopropane ring mostly at C24 or C22 (Figure 3) (3). Based on these characterizations, fossil steranes found in sediments are used as biomarkers for past eukaryotic life (3). The presence of 4-methylsterols in some bacteria shows that some of the necessary biochemical apparatus is present in a few prokaryotes (Figure 4) (7). The use of complete genomes is in fact essential to infer presence or absence of homologs of the different enzymes (3). Phylogenetic trees have shown that the sterol pathway in bacteria and eukaryotes have a common ancestry (7). The native enzymes in plants and fungi, cycloartenol and lanosterol squalene-oxide synthases, are catalytically and structurally similar to the human oxidosqualene-lanosterol and bacterial squalene-hopene cyclases (2). The genomes of several cyanobacteria have been fully sequenced and amongst the genes recognized are several that have a close sequence match to genes related to sterol biosynthesis (7). These include a putative Δ24 sterol C- methyltransferase gene (7).
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Figure 3. Sterols characterized from organisms other than fungi, vertebrates, and land (3).
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Figure. 4. Evolutionary split between prokaryotes and eukaryotes in the biological pathway for their respective membrane sterols (2).
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1.2 Sterol Biosynthesis Recognizing how sterols vary across different species and how they operate in the cell can help lead to the discovery of new means to target harmful microbes and a better understanding on how genetic defects in these biosynthetic pathways can harm the human body. Sterol biosynthesis in plants and fungi differ distinctly from that of animals because they contain more sterol genes over animals (Figure 5) (2). In plants, different genes can encode for similar reaction steps whereas mammals generally have only a single gene for each enzymatic step (2). Among the variety of the sterols, a number of features are in common with the products of these pathways: i) they are all derived from cyclization of squalene into lanosterol or cycloartenol, ii) final products have no methyl group in position 4 and position 14 compared with the first products of cyclization of squalene, iii) they are free of double bonds between C8 and C9, and iv) they present a double bond between C5 and C6 (3). Lanosterol, a common precursor in most organisms, is the product of squalene-oxide cyclization in organisms of a non-photosynthetic lineage while cycloartenol, its homolog in some plants, is the product of squalene-oxide cyclization in organisms of a photosynthetic lineage (2). Lanosterol or cycloartenol is converted to cholesterol in vertebrates, ergosterol in fungi, and to campesterol, sitosterol, and stigmasterol in land plants by succession of oxidations, reductions, and demethylations (Figure 5, 6) (3). Ergosterol and sitosterol are regarded as phytosterols, which are sterols that contain a C24-alkyl group in the sterol side chain, and cholesterol is regarded as a zoosterol, which are sterols that lack a C24-alkyl group in the side chain (2). The fungi pathway is very similar to that of vertebrates, however, the ergosterol produced contains 28 carbons contrary to cholesterol, which only contains 27 carbons; sitosterol and stigmasterol produced by land plants contain 29 carbons (3).
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Figure 5. Generalized synthetic pathway of sterols. Sterol precursor squalene is oxidatively converted to oxidosqualene, which is cyclased to one of two protosterols: cycloartenol or lanosterol. The protosterol undergoes subsequent modifications including oxidative demethylations and desaturations to result in the terminal sterol product. Enzymes are labelled with EC number where available, or gene abbreviation. Terminal sterols yield derived steranes after burial and diagenesis. Enzymes labelled in bold are discussed in the text. Those requiring molecular oxygen are noted (8).
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A 3
4 +CH3 7 Ergosterol SMT
1 2 5
6 8 Sitosterol
B Natural Substrate for C24-SMT Across Kingdoms
H HO 4 O 4 HO 4 HO 4
Lanosterol Cycloartenol 24(28)-Methylene Lophenol Zymosterol 4,4-Dimethyl Sterol 4,4-Dimethyl Sterol 4-Monomethyl Sterol 4-Desmethyl Sterol
Fungi Plants Plants Fungi
Figure 6. The representative phytosterols (24-alkyl sterols) with the different side chains, panel A, that ultimately lead to ergosterol and sitosterol (5). In panel B, the preferred substrates for the C24-SMT.
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1.3 Functional Domain of Sterols Sterols are amphipathic compounds that originate in the isoprenoid biosynthesis, having a main frame composed of a 1,2-cyclopenta(a)perhhydrophenanthrene nucleus of A, B, C, and D-rings (2). Their structure possesses four indispensible domains that make it unique and complementary for membrane insertion: a) Equatorial 3-hydroxyl group, b) Planar tetracyclic ring system also known as the nucleus, c) Right-handed C20R configuration, and d) C8- or C10- side chain (Figure 7) (9). In Domain A, the polarity and tilt of the C3-OH group add functionally to hydrogen-bond interactions while the C4- and 14-methyl groups can affect the A ring conformation and back face planarity, respectively, in Domain B (2). Alternatively, the number and position of double bonds in the nucleus can affect the shape of the sterol and tilt of the 17(20)-bond (5). In Domain C, the natural configuration at C20R determines the conformation of the side chain to orient into a “right-handed” position (2). Variations in the length and orientation of both the C24-alkyl group and the double bonds can be found in the side chain of Domain D (10). These four domains possess strategically positioned chiral carbons that contribute to the polarity and shape of the sterol, producing an alternating all trans-anti stereochemistry of the ring system (10).
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Figure 7. Diagram of the four regions of importance for membrane insertion as illustrated on ergosterol (fungal sterol). Domain 1 is the equatorial 3-hydroxyl group; Domain 2 is the planar tetracyclic ring system also known as the nucleus; Domain 3 is the right- handed C20R configuration; Domain 4 is the C8- or C10-side chain (9).
An organism must maintain the precise 3-D conformation of its specific membrane components that are involved in vital cellular functions (11). The Δ5- phytosterols often constitute < 80% of the total sterol fraction of cells and accumulate in the plasma membrane (12). Double bonds are formed, broken and/or shifted throughout the mechanism in order to achieve the final product for it to be inserted into the
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membrane. These molecular features are crucial for the Δ5 –sterol molecule to function in the membrane as a flat, elongated compound (Figure 8) (2). The principal difference in these sterols lies in the side chain with different degrees of substitution and unsaturation (2). An important distinction for sterol usage in fungi and plants that’s different from those found in mammals is that these microbes must retain the C24-alkyl group in the sterol side chain for the sterol to carry out its individual functions other than that of a bulk membrane insert (13). The 24-alkyl group is preserved during subsequent steroid metabolism in both fungi and plants to give hormones that regulate growth and reproduction in a manner similar to animals (12). The final product, ergosterol, has the addition of a C24β-methyl group while the membrane-inserted sitosterol has a C24β- ethyl group attached to the C24 position in plants (9). The isopentenoid pathway to squalene oxide can bifurcate to generate the start of the sterol pathway which in fungi begins with the formation of lanosterol and in plants begins with the formation of cycloartenol (Figure 9) (12). The direction of carbon flux proceeds forward to produce Δ5-sterols (12).
Figure 8. Conformational perspective of relevant sterols illustrating the flat structure and the tilt of the C3 and C17 (14).
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Figure 9. Diversity of 24-alkyl sterol biosynthesis scheme. The final sterols brassicasterol and stigmasterol can be formed by different routes that occur in protozoa, fungi and plants (13).
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1.4 Sterol Methyltransferase The alkylation step occurs in phytosterols at C24 in the fourth mentioned domain
of the sterol, the side chain, derived by transmethylation from S-adenosyl-L-methionine (SAM) and catalyzed by the enzyme C24-sterol methyltransferase (C24-SMT) (9,15). This is often the rate-limiting step in phytosterol biosynthesis and sets the pattern for sterol specificity and diversity (16). Together these enzymes are responsible for the formation of more than 200 distinct 24-alkyl side chain structures, representing only a small fraction of the estimated natural variation seen in nature (16). Three different sterol C24-methyltransferase enzymes have been detected and classified according to the substrate favored by the enzyme for catalysis. SMT1 will bind to Δ²⁴⁽²⁵⁾-sterols and SMT2 will bind to Δ²⁴⁽²⁸⁾-sterols (Figure 10) (15). Plants synthesize two C24-SMTs, SMT1 which prefers cycloartenol and SMT2 which prefers 24(28)-methylene lophenol (5). Fungi and protozoa synthesize C24-SMT1 of substrate preference for zymosterol
(Figure 11) (5). C24-SMT monofunctional (C1-transfer activity) or bifunctional (C1andC2- transfer activities) enzymes are bound in the endoplasmic reticulum of the cell and utilize two substrates, SAM and sterol, in the construction of the phytosterol side chain (16). The bisubstrate reaction catalyzed by SMT is characterized by a reorganization of at least 3 bonds: i) cleavage of the C-S bond in the methyl donor, ii) formation of a C-C-bond on the sterol acceptor with a 1,2-hydride shift on the opposite face of the substrate double bond, and iii) loss of a proton from the donor or acceptor (15).The ternary complex that is formed by the SMT catalyzed reaction involving two substrates can proceed by two possible routes, random or ordered (12). Yeast C24-SMT functions in a random bi bi method, meaning either the sterol of SAM can bind to the enzyme in any order without the presence of the other, while a plant SMT enzyme from soybean operates in an ordered mechanism with SAM binding first (15,17). In yeast, C24-SMT produces a single product by concerted action, while all other C24-SMTs studied can synthesize products by a step-wise mechanism by means of the production of cations at C24 and C2 (9).
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Figure 10. SMT1 and SMT2 are distinguished by substrate specificity toward the Δ²⁴⁽²⁵⁾- and Δ²⁴⁽²⁸⁾-olefin side chain structure. The gene families are grouped into five distinct subfamilies based on sequence relatedness and substrate preference, as indicated in the figure (5).
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Figure 11. Ergosterol biosynthetic pathway in fungi showing all the necessary steps for the production of a membrane inserted sterol (9).
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The reaction progress is efficiently channeled through a coupled methylation- deprotonation step to produce a single or set of products. A steric-electric plug model for the coupled methylation-deprotonation of Δ24 –sterols to C24-methyl(ene) products has been previously proposed in our lab with the methylation reaction taking place via a nucleophilic attack by the π-electrons of the Δ24 –double bond of various sterols on the methyl group of the sulfonium group of SAM (Figure 12). The reaction can lead to a high energy intermediate (HEI) possessing a 24β-(si-face attack) methyl or ethyl at C24 and a bridged carbenium ion across the 24,25 bond or a transient C25-carbocation (12). After a
1,2-hydride transfer of H24 to C25 (first C1-transfer), an elimination of a proton at C28 occurs, giving rise to a 24(28)-methylene structure (12). A central feature of this stereochemical model is the enzyme recognition of sterol functional groups at either end of the molecule. The equatorial C3-hydroxyl group acts as the polar head while the side chain functions as a non-polar tail. Differences in the anatomy of these flexible substrates target the shape of the nucleus and the ring structure’s influence on the tilt of sterol C3 hydroxyl and the 17(20) –bond (14).
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1. E + S COMPLEX
SMT Enzyme
- B+ B
R H H 1 Si-face S+ H C H R H 2
2. Activated COMPLEX
SMT Enzyme - High Energy Intermediate
H
+ - B C H R H S 1 R 2
Path a Path b Path c Path d
24(28) 25(27) 23(24) 24(25)
C24 Methyl Products
Figure 12. The steric-electric plug model of the sterol methyltransferase binding and catalysis of sterol and AdoMet; SMT: sterol methyltransferase enzyme, E: enzyme and S: substrate. 18 Texas Tech University, Alicia L. Howard, December 2016
1.5 Substrate Specificity and Analogs Understanding active site substrate requirements for sterol biosynthesis has been limited due to the absences of three-dimensional structures of sterol bound to the C24- SMT (14). A non-structural approach that compares active sites of related enzymes on the basis of substrate specificities has been previously proven useful although the factors of substrate binding that governs catalytic orientation of the reactants in the activated complex remains unclear (14). This specificity for substrates allows C24-SMTs to be studied for their discrimination between analogs of the natural substrate that differ by a single functional group (14). It was proposed that directionality is a relevant factor of interaction determined by the crucial non-reacting substrate group at the proximal end of the acceptor molecule at C3-OH (14). A series of lanosterol analogs which systematically deviate from the C3β-OH group were evaluated against the cloned PbSMT in order to gain further insight into the nature of the enzyme-substrate complex of C24-SMTs (Figure 13).
HO 3 H } O } } F H 1 4 7
H H HO } H N } } H 2 2 5 H 8
H H H O + = } H CO } } H N 3 3 CH3CO 6 3 9
Figure 13. The parent structure (1), lanosterol, and the compounds assayed with the varied C3 residues labeled in table 1. 19 Texas Tech University, Alicia L. Howard, December 2016
The sterol A-ring has several non-reacting features that can influence enzymatic activity (14). The natural substrates can possess slightly different nuclear conformations that are affected by the number and location of double bonds, varied C4-substitutions, and a distinct C3-polarity that is associated with either a 3β-OH or 3-oxo group (14). There has been previous work done examining the accessibility of the 3β-oxygen in binding. To gain further understanding on the catalytic role of the polar group at C3, the nature and magnitude of C24-methylation of a series of synthetic lanosterol derivatives that differ in C3-electronics, size, or stereochemistry were examined kinetically with
PbSMT with the specificity constants, Vmax/Km, of the substrate analog compared to the catalytic competence of lanosterol normalized to 100% (Table 1). The polarity of C3 was systematically decreased from that of C3 in lanosterol in the following order: alcohol > ketone > amine > ester > ether > alkane or hydrogen atom with PbSMT recognition of these analogs as follows: C3-hydroxyl (100%), C3-oxo (47%), C3-amino (21%), C3- methyl ether (18%), C3-acetyl (15%), and C3-hydrogen (0%). The binding affinity of the acceptable analogs is comparable to that of the natural substrate (Kmapp = 21 µM) with
differences in Vmax which suggests that substrate specificity is determined mainly by the transition state structure, rather than by that of the ground state structure. The hydrogen atom from C3-hydroxyl group of lanosterol may not engage in hydrogen bonding solely as an H-donor since the C3-amino analog bears an acidic proton at pH 8. These analogs have contrasting polarity to the protonated amine and possess a lone pair of electrons on the oxygen which are available for the nucleophilic interactions such that the oxygen group may accept a proton in hydrogen-bonding interactions.
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Table 1
Catalytic competence of sterols to the PbSMT.
Substrate Structure Vmax / Km Competence % LA-3ß-OH 1 43/21 = 2.04 100 LA- 3α-OH 2 7/23 = 0.30 0 LA- 3β- OMe 3 9/25 = 0.36 18 LA- 3- OXO 4 22/23 = 0.95 47 LA- 3β- Ac 5 25/30 = 0.83 41 LA- 3β- NH2 6 13/30 = 0.43 21 LA -3β- F 7 10/25 = 0.4 20 LA- 3- H 8 0/0 = 0 0 LA- 3β- NH3⁺ 9 0/0 = 0 0
Structures of substrates are given in Figure 13. LA is lanosterol; 100% of competence is normalized to 2.04.
Steady state kinetic analysis of the inactive substrate C3-deoxylanosterol against lanosterol showed competitive-type inhibition (Ki approximately 67 µM) suggesting that the strict regio- and stereo-specificities of the C24-methylation reaction were kept constant with the correct anchoring in the activated complex, but lacked the necessary structural features at C3 to induce the conformational change required for catalytic activity and consequentially does not undergo a reaction. The principle determinant during binding appears to be associated with Domain A and the C3-hydroxyl group, since removal of the C3-hydroxyl group on the analog by replacement with the hydrogen is capable of competitive inhibiting the SMT. The affinity of C24-SMT for its substrates depends on enzyme polar interactions with the C3-hydroxyl group and C24 double bond, and also involves nonpolar interactions of the nucleus and side chain. The molecular features which determine the affinity of transition state analogues should be different from those affecting the recognition of the substrates in their ground state.
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1.6 Research Objections The sterol methyltransferase control governs the C24-methylation pathway with precision to generate single or multiple products. A major determinant that controls product diversity is believed to be the precise conformation of the sterol side chain at the sterol binding site (18). Nes and colleagues have performed previous work on the cloned S. cerevisiae SMT1 in Escherichia coli cells through analysis of the highly conserved motifs of different C24-SMT sequences across kingdoms to explore the three- dimensional structure as well as perform kinetic studies to achieve a better understanding on the mechanism of the coupled methylation-deprotonation reaction that takes place in the active site. Although many of the mechanistic and stereochemical details of the C24- alkylation pathways have been verified, little is still known about the active site of an SMT or the manner in which a SMT enzyme imposes a particular conformation on its acceptor molecule, or how it precisely controls the coupled-methylation deprotonation
reaction and establishes kinetic and substrate specificity for C1 and the successive C2- methyltransfers that ultimately giving rise to the side chains of fungal ergosterol and plant sitosterol, respectively (19). Ergosterol is necessary for the regulation of membrane permeability and fluidity, and for regulating the activity of membrane transporters so due to its indispensable role in yeast physiology, ergosterol and its biosynthesis have become a major target in the development of antifungal drugs over the last 65 years (19). A proper plasma-membrane composition is essential and has to be carefully controlled by the cell so inhibition of the C24-SMT will result in depletion of cellular 24-alkyl sterol membrane inserts accompanied by growth inhibition. Understanding how C24-sterol methyltransferases precisely interacts with its substrate in the reaction pathway to produce its membrane- insert is vital before medical applications against the SMT enzyme can be used to fight fungal infections. The different sterol composition of the plasma membrane plays an important role in short-term and long-term processes that accompany the exposure of stress caused by inhibitors (19). The changes in the yeast sterol profile were analyzed as a function of media supplementation with the antifungal drugs, 25-azalanosterol and abasol, at their IC50 concentrations to improve the understanding of sterol biosynthesis in 22 Texas Tech University, Alicia L. Howard, December 2016 pathogenic fungi studied for antifungal drug development. Characterizing the sterol metabolomes involved in growth and developmental regulation as well as those involved in the state of cellular stress is important so that catalyst-based sterol analogs can be designed to control flux at the rate-determining step of the pathway (13). The investigation of these sterol analog inhibitors along with the work of mutational analyses have provided some information about the size and shape of the enzyme pocket and the identity of amino acids involved with catalysis. In keeping with previous interest in this laboratory with the C24-SMT, the aim of this research was to establish the uniformity and the differences in structural requirements for sterols through growth studies, substrate analogs, and mutagenesis.
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CHAPTER II
EXPERIMENTAL METHODS
2.1 Chemicals and Solvents All chemicals and solvents were purchased from VWR, Fisher scientific international, Sigma-Aldrich, Midscience, Inc (St. Louis, MO), or Promega Inc unless noted otherwise. Bradford protein assay kits were purchased from Bio-Rad laboratories (Palo Alto, CA). IPTG (isopropylβ-D-1-thiogalactopyranoside) was purchased from Boston bioproducts.
2.2 Substrates Sterol sources, unless otherwise noted, were purchased from Sigma Aldrich, in St. Louis, as a crude powder, recrystallized and purified by HPLC. Most Sterols, such as Cholesterol and Ergosterol were purchased around 94-95% pure and were further purified by recrystallization. Other sterols were prepared and synthesized through the Dr. W.D. Nes sterol collection. Sterol compounds were analyzed and quantified by gas-liquid chromatography on an HP5890 chromatograph equipped with a flame ionization detector (FID).The substrate Cholesterol was used as a standard, which was prepped at 1g/L in ethanol. The coenzyme SAM chloride salt, 100% purity, was from Sigma. Abasol was synthesized at Bayer AG, Leverkusen, Germany with the chemical abstracts service (CAS) registry No. 129639-79-8.
2.3 Yeast Culture and Growth Conditions Saccharomyces cerevisiae wild type strain was cultivated to stationary phase on autoclaved medium of 1% yeast extract-2% peptone-2% dextrose (YPD) in double- distilled water. Cell culture was preformed normally with 25 mL of YPD medium in 125 mL Erlenmeyer flasks. Media was inoculated with S. cerevisiae starter culture at the approximate density of 1.0 x 106 cells per milliliter. Cultures were aerobically incubated for 48 continuous hours at 180 rpm and 30 degrees Celsius in a G10 gyrator shaker.
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Growth was monitored every four hours for the first 24 hours post-inoculation and every six hours during the subsequent 24 hours. At each interval, cell morphology was examined under light microscope at 400X magnification with 100 µL of cells diluted in 900 µL of distilled water. The cells were counted with a hemacytometer (Reichert Bright- Line, 0.1 mm) according to the following conditions: Budding cells at least 50% of the parent cell size were counted as separate cells with growth arrest defined as 108 cells per milliliter of medium. The cells per liter were equaled to the average count per square on the hemacytometer multiplied by the dilution factor and then multiplied by 106.
2.4 Analysis of Sterol at IC50 Immediately prior to inoculation with S. cerevisiae wild type, the following concentrations of 25-azalanosterol and abasol were added to YPD culture media via stoichiometric dilution of prepared stock solution (50 nM, 500 nM, 5 µM, and 10 µM in ethanol solvent with a concentration of no more than 2% of the total volume of medium). 25 mL of media was inoculated with a density of 1.0 x 106 cells per mL and the culture was incubated at 30°C for 48 hours while shaking at 180 rpm and then transferred to centrifugal tubes. These were centrifuged at 10,000g for 10 min. The supernatant was discarded and 50 ml of 10% methanolic-KOH was added to the pellet. The tubes were vortexed until the cell pellets were broken up. After vortexing, the mixture was transferred to a 125 mL round bottom flask containing a stir bar and boiled at reflux while spinning for 30 min for saponification of the sterols. The solution was cooled and a 1:1 of distilled water to the methanolic-KOH previously added was added to the round bottom flask. The sterols were then extracted three times with twice the volume of hexane by using a separatory funnel. Hexane was collected and evaporated using a rotovap. The residue was dissolved in acetone. The final non-saponifiable lipid fraction were recovered and an aliquot was injected into the GC-MS to obtain preliminaty sterol quantification and identification.
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2.5 Functional Expression of Cloned C24-SMT Generation of native recombinant SMT (S. cerevisiae, C. reinhardtii, and G. max) was performed as previously described.13 BL21 (DE3) strain E. coli cells containing the SMT plasmid were stored at -80°C in 25% (v/v) glycerol Luria broth medium (LB). Aliquots were transferred from -80°C storage to an agar plate ( Difco bacto-tryptone 10 mg/mL, Difco bacto-yeast 5.0mg/ml, NaCl 10 mg/mL, and agar 15 mg/mL, pH 7.0) containing the preferred antibiotic (ampicillin or kanamycin depending on vector) at 50 μg/mL and incubated for 12- 14 hours at 37°C. A single colony was used to inoculate a 50 mL Luria-Bertani (LB) (composed of Difco bacto-tryptone 10 mg/mL, Difco bacto- yeast 5.0 mg/ml, NaCl 10 mg/mL, pH 8.8) containing an antibiotic concentration of 50 μg/mL. A 125 mL Erlenmeyer flask was used to incubate the culture at 37°C for 12 hours by shaking 225 rpm. To optimize cell activity the optical density at 600 nm from a Beckman Counter DU 530 UV/visible spectrophotometer was measured to be no greater than 1.2. 20 mL aliquots of the starter culture were transferred to a 2.8 L Erlenmeyer flask containing 1 L of LB medium containing antibiotic (50 μg/mL) and incubated at 37°C at 225 rpm until the optical density at 600 nm was measured to be about 0.6-0.7. In order to induce the C24-SMT, isopropyl-l-thio-P-o-galactopyranoside (IPTG) was added to each culture to give a final concentration of 0.4 mM. The cultures are incubated for an additional 3 hours at 30°C at 180 rpm. Cells are then harvested by centrifugation at 10,000g for 10 minutes and either used immediately or stored at -80°C for a maximum of 2 months. The cell paste was resuspended in 20 mM phosphate buffer (0.2 M KH₂PO₄, 0.2
M K₂HPO₄, dilute 5x dd H₂O). and 5 % glycerol (v/v) at pH 7.5. For every gram of cells used, 10 mL of buffer was added. Typically, 2 g of cells in 20 mL of buffer was used to perform a protein assay. The suspension was then lysed by passage through a French pressure (SIM AMINCO) cell pre-cooled to 4 °C at 20,000 psi (equivalent to 1260 units) and then used for determination of products. The assays conducted did not require the use of a pure protein. The lab previously purified the ScSMT and used a suicide substrate to identify the amount of protein in the lysate. Earlier research in the lab used a soluble protein and the results were similar to the crude protein used in this research study. 26 Texas Tech University, Alicia L. Howard, December 2016
2.6 Standard Enzyme Assays A standard assay for C24-SMT enzyme activity was performed in 10 mL test tubes with 600 µL of total volume containing fixed amounts of substrate with a 100 μM concentration for product distribution in 12 μL 5% Tween 80 prepared with 200 proof ethanol (5 mL of Tween 80 dispersed in 95 mL of 200 proof ethanol). The substrate packaged in Tween 80 was pipetted to the bottom of the tube and dried under a flow of nitrogen gas. 2 mg/mL of total crude protein (standard Bradford assay for protein concentration) with enough phosphate, pH 7.5, buffer to bring the volume to 500 µL, was added to the test tube and the reaction initiated with the addition of 100 µM SAM that was prepared in 0.001 N H₂SO₄ to give a concentration of 6 mM and stored at -20°C. A working stock solution was made by a 1:10 dilution of the previous prepared SAM in phosphate buffer pH 7.5 to have a final concentration of 0.6 mM. Incubation was at 35°C in a water bath for 16 hrs with constant shaking. The reaction was terminated with 600 µL of a solution of 10% Methanolic-KOH. After heating at 80°C for 20 mins and allowed to cool to room temperature, the methylated sterol product was extracted three times with 2.5 mL each of hexane, vortexed, and centrifuged for 2 min. The resulting organic layer was dried under nitrogen for determination of substrate conversion and product analysis by GC-MS. GC-MS was conducted on a Hewlett Packard 6890/5973 gas chromatograph/mass spectrometer (70 eV) equipped with a 30-meter Aligent HP-5 5% phenyl methyl siloxane capillary column, internal diameter of 250.00 μM and a film thickness of 0.25μM. The initial oven temperature was 170ºC for one minute with a temperature ramp of 20ºC per minute until a final temperature of 280ºC was reached with an inlet pressure of 113.584 kPa. Chromatograms began recording analyte signals after three minutes.
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2.7 Protein Quantification: Bradford Method The protein concentration was determined by the Bradford method. A calibration curve was generated using incrementing concentrations of bovine serum albumin (BSA, purchased from Sigma as a lyophilized powder) prepared as follows: From the stock solution (1mg/mL) 5 μL to 25 μL were siphoned into 2 mL microcentrifuge tubes. The volumes were made up to 25 μL with double distilled water. To these tubes, 25 μL of 1 M NaOH was added and allowed to stand at room temperature for 20 minutes to denature the protein. The Bradford reagent was prepared by diluting the commercial stock (1:4) with double distilled water before use. After the incubation with 1 M NaOH, 950 μL of diluted Bradford reagent was added to all the tubes and allowed to incubate at room temperature for 10 minutes. A blank was prepared using with 25 μL of water replacing the protein standard. The absorbance at 595 nm of the BSA standard solutions was read against the blank. A linear regression was calculated from the plot of the absorbance at 595 nm as a function of BSA concentration in μg. Solutions of the crude protein were prepared similarly, and their absorbance at 595 nm was measured. Protein concentration was determined using the linear regression of the calibration curve of the BSA standard.
2.8 Preparation Plasmid BL21 (DE3) strain E. coli cells containing the cloned C24-SMT plasmid was transferred to a 50 mL LB culture containing 1% Tryptone, 1% NaCl, 0.5%Yeast extract, pH 7.0 and appropriate antibiotic (50 μg/mL). Incubation was done while shaking at 225 rpm for 16 hours at a temperature of 37 °C. After incubation of the above plasmids at 37°C for 16 h shaking at 225 rpm, the cells were purified using QIAprep Miniprep Kit (Qiagen) or Wizard® Plus SV Minipreps DNA purification system (Promega) as per manufacturer’s instructions. The kits yielded between 50 and 100 ng/μL as determined by the UV-spectrometry absorbance at 260 nm. Unless immediately used, the plasmids were stored at -20 °C.
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2.9 Site-directed Mutagenesis The “Quick-change Site-directed Mutagenesis Kit” (Stratagene) was used as per the manufacturer’s instructions to generate mutations at specific locations. The method consists in first designing oligonucleotides primers with about 15-20 base pairs before and 15-20 base pairs after the target amino acid. Primers were between 30-39 base pairs in length with a thermodynamic melting temperature (Tm) between 60 and 75 °C. The 5’ and 3’ ends of the primer typically end with a Guanosine (G) or Cytosine (C) with a G/C percentage of at least 40%. All primers designed and developed for this study are shown in table 12. To initiate mutagenesis, Polymerase Chain Reaction (PCR) was employed using 50 ng of purified plasmid DNA, 125 ng each of Sense and Antisense oligonucleotide primers, 1 μL of 20 mM dNTP (deoxynucleotide triphosphate) mix, 5 μL of 10X Reaction Buffer, sterile double distilled water made up to 50 μL followed by the addition of 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) in a sterilized 0.2 mL eppendorf PCR tube. The thermal cycling parameters were selected to follow the suggested guidelines, i.e. 1 minute per kb of plasmid length. The parameters followed were therefore: 95°C for 30 seconds, 95 °C for 30 seconds, 55 °C for 1 minute and 68 °C for 6 minutes (for a 6 kb plasmid) repeating the cycle 16 times and then cooling to 4 °C. Finally, 1 μL of Dpn I restriction enzyme (10 U/μL) was added to the amplification mixture to digest the non mutated dsDNA, and incubated at 37 °C for 1 hour. To transfom the plasmid containing the recombinant DNA, competent E coli BL21(DE3) cells (Stratagene, stored at -80°C in aliquots of 50 -100 μL) were used. The competent cells were kept on ice until they were thawed. Once thawed, 100 µl was aliquoted with 1.7 µl of the β-mercaptoethanol to a pre-chilled 14ml BD falcon Polypropylene round bottom tube and chilled on ice for 10 min. About 50 ng of plasmid DNA (1 μL PCR product) was added to the competent cells, gently mixed and allowed to continued incubation on ice for 30 minutes. The competent cells were subjected to heat- shock at 42 °C for 45 seconds followed by incubation on ice for 2 minutes. 900 μL of SOC medium (for 1 L: 20.0g tryptone, 5.0g yeast extract, 0.5g NaCl, 10ml filter- sterilized 1M MgCl2, 10ml filter-sterilized 1M Mg SO4, and 2ml 20% glucose) were added to the plasmid containing competent cells and incubated at 37 °C for 1 hour with 29 Texas Tech University, Alicia L. Howard, December 2016 shaking at 245 rpm and finally plated on LB agar plates containing 50 μg/mL of the appropriate antibiotic. The plates were incubated upside down overnight for 12-14 hours at 37 °C. The colonies were then screened for the point mutation by DNA sequencing performed at the Biotech Core Facility (Texas Tech University) using the T7 sequence as the promoter or the terminator.
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CHAPTER III Saccharomyces cerevisiae Growth Studies
3.1 Previous Research with Fungal C24-SMT The frequency of occurrence of human fungal infections has been increasing over the past few decades with the most prevalently utilized antifungal agents, the polyenes and the azoles, used to treat these infections (20). The polyenes are effective by binding to ergosterol, the fungal membrane sterol, and inducing lethal cell leakage, but often comes with negative side effects and resistance (20). The azoles function by inhibition of the cytochrome P450-mediated removal of the C14-methyl group from the ergosterol precursor, lanosterol (20). Although resistance to polyenes is less common than that to azoles, it is increasingly reported in pathogenic yeasts and filamentous fungi (21). The emergence of antifungal resistance in yeast clinical isolates may have contributed to the increase in the prevalence of invasive candidiasis. The increase in infections coupled with the reduced efficacy of the currently available drugs makes the discovery and development of new antifungals an urgent matter (20). Among pathogenic yeast species, Candida glabrata ranks second in all clinical forms of candidiasis today (21). This opportunistic pathogen is critical in immunocompromised patients with growing concern for the treatment of infections caused by C. glabrata (21). In previous yeast studies with Vandeputte, a missense mutation with a replacement of a guanine with a thymine at position 592 leading to the replacement of a cysteine amino acid at position 198 with a phenylalanine was detected in the C. glabrata sterol Δ24-methyltransferase sequence that caused polyene-resistance (21). Antifungal susceptibility testing with the mutated gene showed a lower susceptibility to polyenes associated with an increased susceptibility to azoles than the wild type (21). The amphotericin B MIC for the mutant was more than 40-fold higher with 1.3 g/mL verses the wild type with a MIC of 0.029 g/mL, whereas azoles MICs were on average 5-fold lower than those for the wild type (21). Chromatographic analysis of the sterol molecular species revealed severe changes in the sterol composition of the polyene-resistant mutant (Figure 14) (21). Ergosterol was 31 Texas Tech University, Alicia L. Howard, December 2016 the major sterol in the wild-type, but was not detectable in the Cys198Phe mutant. In contrast, numerous sterol intermediates identified through UV light absorbance at 281.5 nm as Δ5,7 dienols containing two conjugated double bonds at C5 and C7 accumulated in the cells of both isolates. Therefore, sterol intermediates detected in cells of the Cys198Phe mutant were considered non-ergosterol Δ5,7- dienols. Since ergosterol is the main target of polyenes, resistance may arise from a decrease in ergosterol content or from a complete lack of ergosterol in the plasma membrane as a consequence of mutations in genes encoding some of the enzymes involved in the ergosterol biosynthesis pathway (21).
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Figure 14. Variations in sterol profiles of C. glabrata isolates 21231 (wild type) and 21229 (mutant). Sterols of the heptanic fraction were analyzed by gas chromatography. As highlighted by the dashed line, ergosterol, which was the major sterol species for isolate 21231 (A), was not detectable on the chromatogram of isolate 21229 (B). (C) Percentages of the ergosterol biosynthesis intermediates determined from the corresponding peak areas and retention times (21).
The inner layer of the cell wall appeared thinner for the Cys198Phe mutant than for control cells, suggesting changes in the biosynthesis of its components or in their assembly. Also, growth of the mutant was inhibited at the lowest concentration of calcofluor white, a cell wall-perturbing agent, at 0.1 mg/mL, whereas a concentration of 2 mg/mL of calcofluor white was required to inhibit growth of the wild type. The mutant seemed unable to produce enough ergosterol to supply growth so it adapted by providing 33 Texas Tech University, Alicia L. Howard, December 2016
a sufficient amount of non-ergosterol Δ5,7-dienols to maintain cell viability, which would explain the thinner inner layer of the cell wall and the retractions of the cytoplasm that were observed. The missense mutation that was detected in the ERG6 gene lead to the inactivation of C24- sterol methyltransferase, which in turn lead to the interruption of the ergosterol biosynthesis pathway and, therefore, to polyene resistance. This research has proven the critical role of the C24- SMT in cell proliferation in eukaryotic microbes and suggests the need for the enzyme to be exploited further in the development of antifungal or antiparasitic drugs.
3.2 Sterol Composition The pathway for fungal sterol biosynthesis has provided an excellent target for antifungal development, but there remain additional sites in the pathway that have not been thoroughly investigated (20). Opportunistic fungi that cause infectious diseases, such as candidiasis, produce ergosterol and other C24-alkylated sterols, which are required for parasitic growth and viability (22). The presence of the C24-alkyl chain in ergosterol and the stigmasterols are introduced by the Δ24–sterol SAM-dependent methyltransferase that adds a methylene residue to the unsaturated sterol side chain (22). The sterol methyltransferase gene (ERG6) represents a particularly good target for rational drug design because this step is not found in cholesterol biosynthesis, thus avoiding some elements of possible side effects (20). These differences in sterol metabolism between parasite and host present opportunities for the design and testing of antimicrobial agents which target pathogen-specific sterol enzymes (23). The ergosterol biosynthesis pathway, essentially studied in Saccharomyces cerevisiae, is a complex metabolic pathway with more than 20 enzymes, and the genes that encode them, identified and known today in this yeast (21). In the case of yeast C24- SMT, the full-length ScSMT cDNA of 1.52 Kb encodes a protein of 383 amino acids with a native molecular weight of approximately 172,000 Daltons and a single binding site for sterol and SAM (16). S. cerevisiae, also known as baker’s yeast, is a universal model organism for fungal sterol biosynthesis and drug action that is researched in our lab for enzyme and sterol analysis. Yeast has a well-documented genome that allows the
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study of sterol metabolism and drug response through the biotechnology applications of mutagenesis and protein activity assays. In our research lab, the S.cerevisiae C24-SMT (ScSMT) gene, ERG6, is cloned into Escherichia coli to produce appropriate quantities of the enzyme for research purposes. Since Candida glabrata is genetically closer to S. cerevisiae than other Candida species with their C24-SMT sequences being 81% identical, it’s the preferred fungi to conduct research on that follows up on Vandeputte’s work as well as previous work done in our lab. A Cys198Phe mutant was made in the cloned SMT of S. cerevisiae to test activity (Figure 15). Because of the mutation at Cys198 in C. glabrata inhibiting the SMT, we were expecting to see a drop in activity or none at all. The mutant was inactive, confirming the findings of Vandeputte. Moving on with previous interest in our laboratory with the C24-SMT, research was carried out in S. cerevisiae to establish the uniformity, differences, and structural requirements for sterol catalysis through the testing of antifungal agents for inhibitory effects. Growth studies were performed with different inhibitors either commercially bought or synthesized by our lab to get a better understanding of what interactions are going on in the active site (Figure 16).
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Figure 15. Alignment of sterol C24-methyltransferase amino acid sequence sequences (GenBank accession numbers) from C. glabrata (AAX73200.1) and S. cerevisiae (NP_013706.1). Identical residues conserved in the primary structure are shaded red. The position of the C198F mutant is indicated by the arrow.
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2
1
3
Figure 16. The growth curve of S. cerevisiae over a course of 48hrs (panel 1). The GC- MS analysis of the product distribution of ScSMT wild type (panel 2). The letters over each peak in panel 2 corresponds with the sterols in the table (panel 3).
The fungal SMT gene from S. cerevisiae, designated ERG6 and the best studied for this class of catalyst, encodes the Erg6p with a substrate preference for zymosterol (16). S. cerevisiae wild type without the presence of inhibitors was grown over a course of 48 hrs with an accumulation of erogsterol which was predicted being that ergosterol is the end product necessary for the cell wall to function. This sterol profile was used as the control to monitor the changes inhibitors might have on the accumulation of sterols within the cells. Previous work in our lab established the functional significance of the structural features of yeast ergosterol. The physiological importance of ergosterol to 37 Texas Tech University, Alicia L. Howard, December 2016
fungal-plant/human interactions provide a biochemical paradigm that ergosterol- dependent diseases can be cured or eradicated through the disruption of ergosterol homeostatis (5). The key element of this paradigm is that de novo sterol synthesis and the structural features of ergosterol are important to fungal growth and that a loss of the native ergosterol structure, through blockage of the addition of the C24-methyl group to the intermediate structure, will harm cell physiology (5). The organisms sterol content and/or composition can undergo a marked change as a function of host-parasite interactions. Since the C24-alkyl sterol (ergosterol) to C24-desalkyl sterol (zymosterol) balance, representing the ratio of end product sterol to pathway intermediates, is considered to be a factor in ergosterol-forming disease processes, work in our laboratory as well as elsewhere has focused on the rational design and development of specific inhibitors of the C24-SMT (16). We focused on these growth studies with two inhibitors, 25-azalanosterol and abasol, while establishing a sterol profile through sterol extractions to understand which intermediates are accumulating and if these intermediates are free sterols in the cell membrane.
3.3 Inhibitors Sterol metabolism is an extremely important area of biochemical differentiation between humans and their pathogenic microbes that might be exploited in the development of antifungal or antiparasitic drugs (16). At least 20 metabolic steps are necessary to synthesize such sterols as cholesterol and ergosterol, with some steps involving specific enzymes that differ between mammalian cells and microorganisms. There are several known drugs that interfere with sterol biosynthesis which are used to treat diseases from fungi and protozoans such as high cholesterol in humans and fungal infections (Figure 17) (24). Some of these enzymes have been extensively studied as targets for the development of new drugs that interfere with parasite growth without severe effects on host cells (24). Statins are one of the main classes of the sterol biosynthesis inhibitors (SBIs), which act on the mevalonate pathway by the inhibition of HMG-CoA reductase (24). They have been widely used for cholesterol reduction in humans, however, a drawback of the statins is their effect on the synthesis of isoprenoid
38 Texas Tech University, Alicia L. Howard, December 2016 compounds that are essential for several cellular events (24). Azoles are important inhibitors of C14α-demethylase, and since they are effective against most fungal diseases, they are presently considered to be the most important antifungal compounds in use (24). Four commercially available triazoles are fluconazole, itraconazole, voriconazol, and posaconazole (24). The last class of ergosterol biosynthesis inhibitors comprises the azasterols and abasol, which inhibit Δ24(25)-sterol methyltransferase. Inhibition of this step appears to have high selectivity for fungi and protozoans since this enzyme is not found in mammalian cells (24).
Figure 17. The biosynthesis of ergosterol and cholesterol showing the main steps, the enzymes involved, and the known inhibitors (24).
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Our lab has currently been doing research on 25-Azalanosterol, a substrate analog with a nitrogen in the side chain, and Abasol, an antifungal agent that inhibits the methylene transfer to the sterol side chain from SAM (Figure 18) (Table 2). Both are considered inhibitors of the C24-SMT enzyme. The purpose of designing inhibitors targeted at the C24-SMT is twofold: i) to obtain insight into the mechanism of the C24- methylation reactions, as well as gain information on the active site topography, and ii) to develop leads to disrupt phytosterol homeostasis associated with disease states (16). As a class, the C24-SMT has been difficult to study because they can be recalcitrant to crystallization for X-ray structure determination, thus providing limited opportunity to fully investigate the chemical reactions carried out by these enzymes using classic approaches (16). For inhibitors to be effective, they should bind much tighter than their normal substrates such that their delivery to the target enzyme can occur at concentrations that greatly exceed the affinity constant of the natural substrate (16). The most catalytically favored conformation of the active site is one that does not fit the ground state conformation, but instead is complementary to the transition state of the reaction. The interaction between the enzyme and the transition state exhibits a much higher affinity than the interaction between the enzyme and the substrate. In order for the ground substrate to bind, the enzyme must undergo a conformational deformation that is energetically unfavorable. The function of a catalyst is to lower the energy of the transition state for the reaction so if the transition state stabilization is equated to the binding energy, a stable analog that mimics the structure of the transition state should bind to an enzyme more tightly than the native substrate (Figure 19) (5). The additional energy obtained from this binding stabilizes the transition state and therefore accelerates the reaction. It should bind with a strength of at least three orders of magnitude higher than that of the best substrate for the enzyme (5).
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Native Substrates
Inhibitors
Figure 18. The structures of the inhibitors tested alongside the native substrate they mimic.
A
Substrate High Energy Intermediate Product
B
Substrate High Energy Intermediate Product analog analog analog
Figure 19. Inhibitors tested with the SMT: A. Native C-methylation reaction progress; B. Rationally designed inhibitors (25).
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Table 2 Sterol composition of Saccharomyces cerevisiae.1
Inhibitor Sterol Time (min) % of total Control Zymosterol 15.45 4.47 Ergosterol 15.96 52.28 Fecosterol 16.76 15.91 Δ7-Fecosterol 17.51 13.79 25-AzaLA Zymosterol 15.51 23.41 Δ5,7,24-Cholestatrienol 15.87 23.53 Δ7-Zymosterol 16.13 16.53 Δ5,7,22,24-Cholestatetraenol 16.52 36.53 Abasol Zymosterol 15.45 12.51 Δ5,7,24-Cholestatrienol 15.81 19.13 Δ7-Zymosterol 16.12 18.52 Δ5,7,22,24-Cholestatetraenol 16.51 41.36
1Percentage of the sterol composition with the time each sterol was eluted from the GC from Yeast wild type grown in 25 mL of media for 48hrs at 30°C 180 rpm with inhibitors at 100 µM.
3.4 25-Azalanosterol
The first generation derivatives with a sterol nucleus and modified side chain considered to be high energy intermediate analogs against natural substrates were designed to mimic the C24-methyl, C25 cationic intermediate (16). These high energy intermediate (HEI) analogs, also referred to as transition state analogs, were prepared by replacing C25 with a nitrogen that can acquire charge as a result of being protonated under physiological conditions (16). A positively charged nitrogen atom introduced at C25 is expected to mimic the structure of the putative “charged” intermediate formed during catalysis (5). The ammonium analogs were prepared with the expectation that the SMT in the ground state would recognize the HEI mimic; the assumption being that the active site binds the C25 nitrogen substrate sterol to the same active center as the substrate and the enzyme will undergo a conformational change during catalysis to recognize the inhibitory group of the substrate analog (12). Inhibitors that contain a
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nitrogen function around C24-C25 usually impair catalysis between 1 and 50 nM (5). However, as the nitrogen is moved closer to C20, the inhibitor becomes less potent (5). The enzyme recognizes the inhibitor as reversible tight-binding analogs that inhibit the
enzyme with a Ki in the low nanomolar concentrations. With the yeast SMT, the Km for zymosterol observed in our lab is 35 µM whereas the transition state analog and potent
noncompetitive inhibitor of the C-methylation reaction, 25-azalanosterol, generated a Ki value of 45 nM.
25-azalanosterol showed a range of antifungal activities, with an IC50 value 1 µM
(Table 2, 3). At the IC50 of the inhibitor, the sterol composition of the treated cells was found to be dramatically modified with the bulk of the ergosterol replaced by zymosterol, the native substrate of the SMT1 (Figure 20). The effects of 25-azalanosterol also produced two new sterol compounds: cholesta-5, 7, 24, trienol and cholesta-5, 7, 22, 24 tetraenol with the disappearance of other compounds (Table 2). These two sterols appeared to be the Δ5,7-dienes detected in cells of the Cys198Phe mutant that lead to the inactivation of C24- sterol methyltransferase from Vandeputte research (Figure 21). After an increase of the inhibitor, it is clear that C24-methyl sterols are essential for the maintenance of membrane structure and function. Fungal growth inihibition by low levels of 25-azalanosterol was caused by the inihibition of sterol biosynthesis. It must be noted that the values of amount of sterol per cell do not correlate well with the IC50 values. At
concentrations below the IC50 values, there was evidence of a loss of ergosterol with an accumulation of zymosterol. This indicates that at low levels of 25-azalanosterol, sterol biosynthesis is already being inhibited with the attempt to use zymosterol and/or its derivatives for membrane inserts. Increased membrane permeability by these substitute inserts can lead to depolarization and loss of proteins, nucleotides and other compounds (26).
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Table 3 Antimicrobal activity of 25-azalanosterol against S. cerevisiae.1
25-azalanosterol
Cell count per Cells/mL per data set data set Control [μM] 1 1 cells/mL @ % growth 48hrs 0.05 325 1.63E+08 1.60E+08 101.80% 0.5 290 1.45E+08 1.60E+08 90.63% 5 4 2.00E+06 1.60E+08 1.25% 10 2 1.00E+06 1.60E+08 0.63%
1See materials and methods for growth conditions
1 2
µM
Figure 20. The sterol composition in panel 2 at the IC50 of 25-azalanosterol (panel 1) with peaks A) zymosterol (32.5%), B) Δ5,7,24-cholestatrienol (21.5%), C) Δ7-zymosterol (10.3%) and D) Δ5,7,22,24-cholestatetraenol (35.7%). Sterol fractions were analyzed by gas chromatography.
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2 1 2 3 4 A 1
1 B
4
3
2
Figure 21. Comparison of the sterol profile of yeast wild type (panel 1-A) against yeast incubated with 10 µM abasol (panel 1-B) with that of C. glabrata isolates 21231 (panel 2- A, wild type) and 21229 (panel 2-B, mutant). Sterol fractions were analyzed by gas chromatography. Both panel A’s show erogsterol as the major peak, which was no longer detectable with an increase of zymosterol in the mutant and the yeast incubated with abasol (panel B’s). Peak 2 and 4 in panel 1-B appears to correspond to peak 4 and 6 in panel 2-B as cholesta-5,7,24-trienol and cholesta-5,7,22,24-tetraenol, respectively, as the Δ5,7-dienols detected in the 21229 isolate .
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3.5 Abasol Abasol is an analog belonging to a new class of microbiocidals that has a chemical structure different from existing antifungals, including azoles, which favors the structure of S-adenosylmethionine SAM, the cofactor (26). The interesting thing about abasol is that it’s a commercial drug that isn’t an substrate or transition state analog, but is an analog of the cofactor SAM. There’s currently only one study on Abasol by Borelli from Germany that claims it has better antifungal activity than all standard azole compounds against Candida and Aspergillus. Abasol reduced the growth rate of C. albicans to 30 and 10% at 0.1 and 1 µg/mL, respectively, after a 24hr period with a complete inhibition observed with 10 µg/mL after a 48hr incubation. Mode of action studies with Abasol started with observations that this agent exerts its antifungal activities regardless of whether the pathogens in question are growing or rather in a resting state (26). The impairment of the cell membrane function was tested by direct measurements of conductivity increase. Studies by Borelli observed abasol against C. albicans caused a release of ions from cells within 2 min after addition of 10 mg/mL or higher concentrations. The ion release was measured as conductivity increase in the supernatant of resting cells. Within this short period, K+ ions were released into the supernatant causing lethal direct membrane damage. The leakage of essential cell components becomes a lethal event only if the cells are unable to compensate for the membrane damage (26). An accumulation of lanosterol was detected indicating inhibition of the C24-SMT and proved that sterols without C24-alkyls are not sufficient substitutes 14 for membrane stability and function. When the incorporation rate of L-[methyl- C]- methionine into sterol in the presence of abasol was examined, it was found that abasol inhibited incorporation of radioactivity into sterol at 0.01 µg/mL or higher. With this information, Abasol was tested against S. cerevisiae to verify the inhibition affects on growth and sterol composition. Abasol reduced the growth rate of S. cerevisiae to 50% and 1% at 6 and 10 µM, respectively, after a 48-hour incubation with a complete inhibition observed with 10 µM of the inhibitor (Table 4) (Figure 22). The sterol composition of the treated cells, like 46 Texas Tech University, Alicia L. Howard, December 2016 with the treatment of 25-azalanosterol, was also found to be dramatically modified with the bulk of the ergosterol replaced by zymosterol which is different from the accumulation of lanosterol observed from Borelli (Figure 21). The effects of abasol also produced cholesta-5, 7, 24, trienol and cholesta-5, 7, 22, 24 tetraenol with the disappearance of other compounds. The inhibition from abasol lead to a depletion of the
C28-sterols that are normally found in fungi and was replaced by C27-sterols that are found in mammals. Sterols without a C24-alkyl group appear to be good substrates for the next step in the fungal biosynthesis with enzyme C8-isomerase, which goes on to produce sterols with a Δ7 and Δ5 nucleus that can be incorporated into the cell membrane because of the lack of ergosterol. However, these substitutes can cause thinning of the cell membrane, which was observed from Vandeputte research, and can eventually lead to cell death. These results indicate that yeast can use zymosterol and its derivatives as substrates to compensate for the absence of endogenous sterols but results in a weakening of the cell membrane. The impairment of the cell membrane correlates with the loss of viability.
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Table 4 Antimicrobal activity of abasol against S. cerevisiae.1
Abasol
Cells/mL Cell count per data per data set set Control [μM] 1 1 cells/mL @ % growth 48hrs 0.05 309 1.55E+08 1.60E+08 96.88% 0.5 301 1.51E+08 1.60E+08 94.38% 5 277 1.39E+08 1.60E+08 86.88% 10 150 7.50E+07 1.60E+08 46.88%
1See material and methods for growth conditions
1 2
µM
Figure 22. The sterol composition in panel 2 at the IC50 of abasol (panel 1) with peaks A) zymosterol (31.3%), B) Δ5,7,24-cholestatrienol (18.7%), C) Δ7-zymosterol (10.5%) and D) Δ5,7,22,24-cholestatetraenol (39.5%). Sterol fractions were analyzed by gas chromatography. 48 Texas Tech University, Alicia L. Howard, December 2016
Previous literature as well as my research has reported antifungal inhibitory effects in the nM and µM range for 25-azalanosterol and abasol, respectively, however, there are concerns on if the inhibitors are fungicidal or fungistatic (4,5). The problem with fungistatic is that they inhibit growth causing a resting state, but do not necessarily kill the fungi (fungicidal). An accumulation of toxic intermediates along with the loss of ergosterol in the ergosterol biosynthetic pathway are believed to cause fungicidal effects. Biochemical analyses indicate that in the case of sterol biosynthesis it’s possibly better to use combination therapy to inhibit more than one step in order to completely eliminate all the sterol substrates which participates in essential reactions (24). Combination drug therapy, the simultaneous administration of multiple antimycotics at sub-inhibitory concentrations for increased pathogenic cell death, could prove to be vital to overcoming common health problems like toxic side effects, reduced efficacy and resistance to currently available drugs. Knowing the full inhibitory effects will allow future decisions of which two combined will have the best outcome of killing the fungi. From the research observed with 25-azalanosterol and abasol, there’s a possibility that the combination of the two and other inhibitors that our lab possesses could not only stop growth, but also kill the cells.
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CHAPTER IV
Mutagenesis of the Sterol C24-methyltransferase
4.1 ATP Binding Region Over the past decade, there have been several advancements and discoveries in the study of SMT enzymes. The SMT genes from several plants and fungi have been cloned, sequenced and expressed in bacteria or yeast and bioengineered into tobacco or tomato plants (25). Although the wild-type SMT is generated in low amounts as a microsome-bound or partially purified system, a recombinant SMT from Saccharomyces cerevisiae has been overexpressed in E. coli in large amounts to permit detailed kinetic and structural analyses (25). The enzyme is membrane-bound and it can distribute among different subcellular fractions, including the endoplasmic reticulum, mitochondria and a "floating lipid layer” with a turnover number of 0.01/s and an isoelectric point of 5.95 (25). From observation of the primary sequence, it was found that the SMT from the yeast Saccharomyces cerevisiae has 383 amino acids with a molecular weight of 43 kDa as a monomer (Figure 23) (18). However, the Nes lab, through protein purification and gel permeation, found that the C24-SMT was indeed a homeotetramer with a gross molecular weight of 172 kDa with four identical subunits, and each containing a single active site for sterol and SAM (25). Generally speaking, a tetramer protein can provide the opportunity to exhibit cooperative interaction between the four subunits which means it could be allosterically modulated.
Figure 23. The primary sequence of ScSMT, accession number gi|6323635
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Previous work from the Nes lab with the purified SMT enzyme generated kinetics in which velocity versus substrate curves relative to zymosterol and SAM were sigmoidal rather than hyperbolic, indicating enzyme cooperativity among the subunits (25). Enzymes that demonstrate cooperativity are defined as allosteric, meaning that the binding of the substrate at one binding site affects the affinity of other sites for their substrates. Knowing the significance of ATP as a signaling molecule in metabolic pathways such as glycolysis, ATP and other signaling compounds were tested as effectors for the SMT in the sterol biosynthetic pathway (27). Studies with ergosterol and ATP as possible effectors of SMT activity, indicated that ergosterol can down-regulate SMT with a competitive type pattern of inhibition with respect to zymosterol (Ki = 65 μM) and that ATP at a concentration of 400 μM, a physiological concentration for yeast, can up-regulate (activate) SMT activity by threefold compared to the specific activity of the control experiment without ATP (27). Without the presence of zymosterol and SAM, ATP was bound by the enzyme in the concentration range similar to the preferred substrates with a Kd of 4 µM. ATP also showed no effect on the equilibrium binding of the sterol to enzyme (27). If there’s an ATP effect, then there must be an ATP binding site. With that in mind, looking back at the ScSMT primary sequence, then there should be an ATP region that is possibly conserved within the sequence. The sequence GXXXXGKT/S, popularly known as Walker motif A, is widely believed to be the site for binding nucleotides in many proteins (28). Since it was founded in 1982 by Walker and colleagues, this sequence has been found in many proteins that bind nucleotides and thereby has gained predictive value for nucleotide binding site in proteins (28). When comparing the Walker sequence with the yeast SMT sequence to identify an ATP binding region, the amino acids GLVAGGKS was found which, indeed, lines up with the Walker GXXXXGKT/S (Table 5). In an earlier set of studies from the Nes lab, 52 amino acids from the S. cerevisiae were mutated to leucine as a screen for a change in activity to identify possible essential residues (29). Some of these amino acids that were screened fell into the ATP region of the primary sequence that resulted in a loss of activity (29).
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Table 5 The Walker sequence across kingdoms.
SMT source Walker Sequence Parent walker sequence GXXXXGKT/S S. cerevisiae GLVAGGKS P. brasiliensis CLVKGGEM C. reinhardtii SL I Q GGES G. max1 GLVEGGKR G. max2 YLTRGGDS T. brucei NLVKGGEL T. cruzi GLVSGGES
With the discovery of a couple of these amino acids being essential, the goal was to first mutate a select few amino acids in the Walker sequence to confirm what was previously published. After mutating Gly347, Val349, and Gly352 to leucine, my results confirmed what was reported from the Nes lab. The next goal was to compare the Walker sequence across kingdoms to look for conserved residues and identify any changes from the amino acids that could potentially be proven as essential. Upon comparing the Walker sequence across kingdoms, it was observed that the first Gly residue varied among the SMTs. The question was then asked if the non-conserved amino acids can be replaced with a related residue of another SMT and the resulting substitution shown to be functionally important to SMT activity so the aim was to change the Gly residue in yeast to the varied amino acids across kingdoms: Asn (N) in Trypanosoma brucei, Ser (S) in Chlamydomonas reindardtii, and Tyrosine (Y) in Glycine max SMT2 (Figure 24,25,26). With the idea that differences within the sequences have yielded divergence, we were hoping to find a change in product ratio or in substrate specificity. Along with screening for substrate/product changes, the amount of activity was also being observed based on the rationale from protein purification data with the Q sepharose column chromatography (Table 6).
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B 1 2
Detector Response Detector Response Detector
A
Time (min) M/Z
Figure 24. In panel 1 is the GC-MS of the ScSMT wild type (control) conversion of cycloartenol to 24(28)-methylene cycloartenol. In panel 2 is the mass spectra of the product peak labeled B eluding in panel 1. The substrate peak is labeled A.
1 B 2
A Response
Detector Response Detector Detector
Time (min) M/Z
Figure 25. In panel 1 is the GC-MS of the ScSMT G347N conversion of cycloartenol to 24(28)-methylene cycloartenol. G347N represents the category: 40% or greater resulting in sigmoidal behavior with an ATP affect. In panel 2 is the mass spectra of the product peak labeled B eluding in panel 1. The substrate peak is labeled A.
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A 1 2
Detector Response Detector Detector Response Detector
B
Time (min) M/Z
Figure 26. In panel 1 is the GC-MS of the ScSMT G347N conversion of cycloartenol to 24(28)-methylene cycloartenol. G347N represents the category: 20-30% of activity resulting in sigmoidal behavior with no ATP affect. In panel 2 is the mass spectra of the product peak labeled B eluding in panel 1. The substrate peak is labeled A.
Table 6 Catalytic competence of S. cerevisiae mutants.1
YSMT mutant Codon Activity 100% G347L CTT 19 G347V GTT 80 G347A GCT 50 G347N AAT 74 G347Y TAT 100 G347S AGT 19 V349L CTT 47* A350L CTC 100* G351L CTG 90* G352L CTT 5 K353L CTG 56* S354L CTC 100*
1See materials and methods of enzymatic assay. *Previous results published in Biochimica et Biophysica Acta 1781 (2008) 344-351.
It was previously reported that activity from using lysate to the protein post Q (the removal of ATP), there was a tremendous drop in activity, roughly 30% active, with no 54 Texas Tech University, Alicia L. Howard, December 2016 real affect on sigmoidal behavior. The activity of the mutants were thus screened to observe which exhibited sigmoidal behavior based on the following guidelines: 5% or less resulted in a “kill” with no activity, 20-30% resulted in sigmoidal behavior but with no ATP affect, and 40% or greater resulted in sigmoidal behavior with an ATP affect. There were no changes in substrate specificity or the product profile with changes in activity ranging from 5 to 100% in activity (Table 6). Gly352Leu was the only one found to be an essential amino acid that’s absolutely conserved across kingdoms in the primary structure that lost activity, which could be due to the change in size. Because previous work showed that a loss of ATP resulted in roughly 30% of activity, it can be assumed that Gly352 is not directly involved in binding ATP but possibly interfering with the backbone residues of another subunit changing the overall confirmation. A lab colleague was then given the ATP mutants to perform further analysis. Using the same rationale, mutants were also made to the first amino acid in the Walker sequence from C. reinhardtii and G. max SMT1 with the same interest in substrate specificity and product ratio from the effects of the mutagenesis. Results were similar to those of ScSMT with changes in activity observed ranging 3 to 89% (Figure 27,28) (Table 7).
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1 2
Figure 27. In panel 1 is the GC-MS of the CrSMT wild type (control) conversion of cycloartenol (peak A) to cyclolaudenol (peak B) and 24(28)-methylene cycloartenol (peak C). In panel 2 is the mass spectra of the product peaks labeled B and C eluding in panel 1.
Figure 28. In panel 1 is the GC-MS of the GmSMT1 wild type (control) conversion of cycloartenol (peak A) to 24(28)-methylene cycloartenol (peak B) and a triplet of 24-ethyl olefins (peak C). In panel 2 is the mass spectra of the product peak labeled B eluding in panel 1.
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Table 7 Catalytic competence of C. reinhardtii and G. max mutants.1
Mutant Codon Tm GC% 2Activity C. reinhardtii Cycloartenol Zymosterol S360L CTG 72.1 °C 66.7% 3 3 S360G GGG 73.3 °C 69.7% 60 120 S360Y TAC 70.4 °C 63.6% 50 68 S360N AAC 70.1 °C 63.6% 55 40 G. max SMT1 Cycloartenol *24(28)-Methylene cycloartenol G341L CTA 64.8 °C 54.5% 45 0 G341S TCT 64.6 °C 54.5% 60 0 G341Y TAC 64.7 °C 54.5% 64 0 G341N AAC 65.0 °C 54.5% 65 0
¹See materials and methods of enzymatic assay. 2Activity percentage is based from wild type SMT tested with substrate as 100% competence. *24(28)-methylene cycloartenol produced no activity in the wild type. Melting point and GC% content of the primers used are given.
4.2 SMT Sequence Comparison and Mutagenesis To date, several classes of SMTs from plants and fungi have been reported in the GenBank (30). It was not until the ERG6 SMT gene from Saccharomyces cerevisiae had been cloned, sequenced and then made available to investigators in the early 1990s was there a serious search for SMTs from other fungi and plants (25). At least 19 different sequences of SMTs from 16 species of plants and fungi have been reported in the GenBank and the structures 1 of 9 related SAM-dependent methyl transferases have been solved (27). The common three-dimensional structure of SAM-dependent enzymes is reflected further in the amino acid sequence motifs that are conserved among a large number of SAM-dependent methyltransferases, including SMTs (27). The predominant SAM binding motifs have been shown to be approximately on the same position on the proteins and at comparable integrals (27). The motif related to the SAM binding region spans across amino acids 124 to 133 in the yeast SMT and is conserved throughout a variety of methyhransferases from many different organisms. To locate additional
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catalytically relevant residues and regions of the SMT, the sequence alignments of SMTs (Figure 29) were used alongside mechanistic reasoning that involved chemical affinity labeling and site-directed mutagenesis of select histidines, acidic residues and an aromatic residue considered important to C28 and C27 deprotonations or cation-π stabilizations that can occur during the reaction progress (29). These efforts led to the determination of signature motifs within the sequence indicative of the SMT within the class of SAM-dependent methyltransferases (29).
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SMT enzyme Identity % Similarity % Saccharomyces cerevisiae 100 100 Chlamydomonas reinhardtii 46 59 Glycine max SMT1 42 58 Glycine max SMT2 41 58 Trypanosoma brucei 42 59
Figure 29. The C24-SMT sequence alignment across kingdoms (GenBank accession numbers) from S. cerevisiae (gi|6323635), C. reinhardtii (gi|15965129), G.max SMT1 (gi|351725990), G. max SMT2 (gi|242755468) and T. brucei (gi|70832598). The five conserved regions are boxed in green.
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Although the reaction pathways involved in sterol C-methylation have been studied, little is known about the active site topography of any SMT or the manner in which a SMT enzyme imposes a particular conformation on its amphiphilic substrate, precisely controls the resulting methylation-elimination reaction and establishes the binding order. With a P.brasiliensis C24-SMT, it was proven that productive binding required the sterol substrate to have a C3β-hydroxyl group along with a planar nucleus, an intact side chain with the length of the native lanosterol, a C20R configuration, and a Δ²⁴-double bond (31,5). The sterol molecule is predicted to bind to the donor site of SMT enzyme through a combination of polar and hydrophobic interactions (Figure 30). Specific amino acids side chains are poised in exactly the right places to aid in the catalytic process itself. These side chains are acidic or basic groups that can promote the addition or removal of protons with most enzymes operating through acid-base catalysis in which protons are transferred between donating or accepting atoms on the substrate and key basic and acidic side chains in the enzyme active site (32). It is tempting to speculate that subtle differences in the amino acid composition of active site residues may alter product specificities exhibited by these enzymes (29). The focus was to then understand the similarity and differences in substrate acceptability and reaction pathways to give C24-methyl and C24-ethyl sterols in the SMTs and to determine the type, number and location of amino acids in the active site that control product diversity.
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Figure 30. Proposed aspects of substrate (A) intermediate (B) and product (C) binding to the PbSMT active site. For lanosterol binding in A, the enzyme presents a binding site that is sterically and electronically complementary, to which the substrate becomes anchored at its C3-hydrophilic group. The sterol C3–OH group interacts in a pre- organized active site with contacts that form hydrogen bonds against the 3-oxygen atom (from a main frame moiety, M) and hydrogen atom of the 3-oxygen atom (from a basic amino acid, B1) forming a hydrogen bonded network to stabilize the ground state structure at the proximal end of the acceptor molecule and the side chain assumes a pseudocyclic conformation. Productive orientation of the substrate side chain affords backside (SN2) addition of “methyl cation” from S-adenosyl-l-methionine (represented by the catalytic sulfur atom, S) to the Δ24-bond generating the C24β-methyl C25 cation shown in B. Deprotonation of the C28 methyl group from a basic amino acid (B2) can lead to the C24(28)-methylene product shown in C, followed by disassociation of the methylated sterol from the enzyme (14).
Consistent with their large overall sequence similarity, C24-SMTs possess five highly conserved regions, three of which contribute to the sterol binding pocket, one of them contains an SAM binding pocket and one involved with ATP binding (16). Due to the relationship between the conserved motifs in SAM-dependent methyhransferases and the related structure of the protein, site-directed mutagenesis is a viable tool to probe the structure-function relationships in the active site of the yeast SMT (Figure 31) (27). Mutants of the yeast SMT have been helpful in understanding the importance and role of these conserved regions. In the absence of a three-dimensional structure of this class of 61 Texas Tech University, Alicia L. Howard, December 2016
catalyst, topology mapping by means of mechanism-based inactivators, photoaffinity probes and directed-mutagenesis have been undertaken by several groups to determine the general characteristics of the C24-SMT active site (16). The nature and arrangement of the amino acid residues in the active site of individual C24-SMTs is crucial to the outcome of the reaction (16).
Figure 31. The general mechanism of site-directed mutagenesis with the mutated codon highlighted in pink and the parent codon in blue.
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4.3 The Significance of Tyr81 Mutants In the case of single amino acid replacements directed at the ScSMT, amino acid substitution of residues scattered throughout the primary structure have been shown to convert the activity of the fungal enzyme to plant-like activities (16). Comparison of the protein sequences deduced from cDNA for diverse SMTs of protozoa, fungi and plants revealed a highly conserved region rich in aromatic amino acids, referred to as Region I, containing a signature motif Y81EYGWGSSFH not present in other SAM-dependent methyltransferases and a L124DVGCGVGGP sequence labeled Region II that is significant in SAM binding (34). The bulk of the conserved residues of Regions I and II are primarily hydrophobic and Region I contains a high percentage of aromatic amino acids which are in close proximity to Region II (15). Thus, it was decided to probe conserved amino acids in Regions I and II that when mutated would disrupt zymosterol and/or AdoMet binding. If Region I is definitively the sterol binding site then it is reasonable to predict that a mutation of a critical residue that is absolutely conserved would disrupt sterol binding and likewise a mutation of a critical residue would disrupt SAM binding in Region II. Aimed at reshaping the enzyme specificity and mechanism, our lab modified the signature motif region I in yeast SMT generating a Tyr81Phe mutant that resulted in a gain-in function. The electron-rich aromatic side-chains from tryptophan, tyrosine, and phenylalanine are essential to proton shuttling and catalysis (15). As each of the other enzymes recognizes a Δ24-sterol acceptor, the aromatic rich domain of Region I have been proposed to be involved in catalysis by providing residues that stabilize intermediate cations generated during the C-methylation reaction (33). A site-directed mutagenesis experiment was performed for the presumptive active site aromatic amino acid Tyr81 because it was hypothesized to contact a nearby transient C25 carbocation intermediate in the activated complex (33). Previous work from our lab changed the structure of Tyr81 to Phe, Trp, Ile, Leu, Val, and Ala by direct substitution followed by assay of the resulting mutants and wild type Erg6p with different substrate analogs to investigate the role of the aromatics and aliphatics of different sizes in the C-methylation reaction (33). It was found that substitution of Tyr81 with phenylalanine, an aromatic residue considered to 63 Texas Tech University, Alicia L. Howard, December 2016
contribute to electron density to catalysis, led to altered substrate specificity affording a bifunctional enzyme capable of two sequential, mechanistically different C-methylation reactions with zymosterol as the primary substrate (33). The mutant performed like a plant SMT by catalyzing the transfer of two methyl groups from SAM to the sterol Δ24- bond (34). With fecosterol as the substrate, the Tyr81Phe generated three sterols: 24E and Z-ethylidene cholest-8-enol and 24β-ethyl cholest-8,25-enol showing different proton elimination paths in the formation of Δ24(28)- and Δ25(27)-olefins compared to the catalysis of the wild type that generated a Δ24(28)-olefin exclusively. The family of SMTs is considered to be a group of homologous enzymes derived from a common ancestor and are therefore structurally related (35). Chlamydomonas reinhardtii is a primitive green algae and is believed to be family of the last eukaryotic common ancestor. The analyses of the amino acid sequence of SMTs annotated in the GenBank suggest that the genome of green algae may be unique among primitive organisms and contains a single SMT2 gene bifunctional in substrate recognition (35). Since the Tyr81 residue was shown important for stabilizing the cationic intermediate during the C-24 methylation reaction, for proper orientation of the substrate side chain to ensure formation of the correct product outcome and for being a vital amino acid in yeast and soybean SMT proven from previous work in our lab, the equivalent mutant was made in CrSMT (35). To confirm existing knowledge and mimic the Tyr81 mutants made in Erg6p, the equivalent amino acid in CrSMT (Tyr110) undergone site-directed mutagenesis experiments changing tyrosine to Phe110, Trp110, Leu110, Val110, Ala110, and Gly110. Each mutant was then tested with the substrates cycloartenol, 24(28)-methylene cycloartenol, and zymosterol (Table 8). Substitution of Tyr110 residue in CrSMT1 by Phe showed that the removal of the OH group from tyrosine had little effect on catalysis which was disappointing since in related studies of fungal and plant SMT1, the Tyr to Phe mutation afforded a gain-in-function that produced plant-like second methylation of fecosterol or unusual product ratios from methylation of 24(28)-methylene cycloartenol. As was expected, the aliphatics showed a strong decrease in product when tested with cycloartenol ranging from 30% to 1% conversion. Tyr110Phe produced similar results to 64 Texas Tech University, Alicia L. Howard, December 2016
the wild type as did Tyr110Trp but the Trp mutant exhibited some channeling with a change in product ratio: 45:55 when incubated with cycloartenol, favoring the Δ25(27)- olefin instead of the preferred Δ24(28)-olefin found in the wild type. The inactivity of all five mutants except for Tyr110Phe toward 24(28)-methylene cycloartanol strongly indicates that a change in hydrophobicity can also lead to a local deformation of the active site pocket allowing a change in the side chain of the products. The aliphatics leucine, valine, and alanine all produced a dramatic product: product ratio different from the wild type when tested with zymosterol with convergence ranging from 55% to 5% that also favored the formation of Δ25(27)-olefins. The results demonstrate that the sterol methyltransferase has been redesigned during evolution to accept different substrates and catalyze variant product specificities. It evolved from changes to modifications in enzyme-substrate interactions that control substrate channeling of the C24-methylation pathway (22). It would appear that a change from a loose to tight arrangement of enzyme- substrate interactions or from a non-polar/neutral to a polar/charged amino acid can influence the positioning of the flexible side chain relative to the deprotonating base affording a switch in product specificities of the C24-methylation reaction (22).
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Table 8 Catalytic competence of C. reinhardtii Tyr110 mutants.1
C. reinhardtii % Conversion P:P Tm GC% CA, MCA, Zymo CA, MCA, Zymo Tyr110Phe 45, 60, 43 65:35, 95:5, 30:70 67.0°C 57.5% Tyr110Trp 65, N/A, 75 45:55, NA, 10:90 68.4°C 60.6% Tyr110Leu 10, N/A, 55 65:35, NA, 55:45 67.9°C 60.6% Tyr110Val 30, N/A, 15 65:35, NA, 60:40 68.1°C 60.6% Tyr110Ala 10, N/A, 5 70:30, NA, 60:40 69.8°C 63.6% Tyr110Gly 1, 1, 1 55:45, NA, 35:65 69.8°C 63.6%
¹See materials and methods of enzymatic assay. The percent conversions and product: product ratio from Tyr110 mutants incubated with cycloartenol (CA), 24(28)-methylene cycloartenol (MCA), and zymosterol (Zymo), as well as melting point and GC content of the primers used. 24(28)-methylene cycloartenol:24β-ethyl cycloart-25(270-enol ratio when incubated with MCA. Cyclolaudenol:24(28)-methylene cycloartenol ratio when incubated with CA. Ergosta-8,25(27)-dienol:ergosta-8,24(28)-dienol ratio when incubated with Zymo.
4.4 Acidic and Histidine Residue Mutations A second mutant in yeast SMT, generated in region I at position Glu82, was also 24(28) found to perform like a plant SMT catalyzing in the first C1-transfer reaction a Δ - as well as a Δ25(27)- olefin product (34). The Glu82 residue in the wild-type enzyme is not likely to be ionized so there is a possibility that some other acidic residue was playing dual roles in the active center, acting as a base, or direct reaction channeling. This prompted the consideration of mutating the remaining highly conserved acidic residues in the wild-type (34). Information regarding the most likely amino acid units for substrate recognition at C-3 and Δ24 and in the catalytic cycle involving deprotonation of C-28 was obtained from site-specific mutagenesis of aspartate and glutamate residues of the enzyme that are conserved in 16 different species that cross evolutionary lines from fungi to plants (34). Replacement of the nine conserved acidic residues at Glu108, Asp125, Asp152, Asp189, Glu195, Glu209, Glu224, Glu246, and Asp276 located towards the N- 66 Texas Tech University, Alicia L. Howard, December 2016
terminus by sterically conservative leucine residues that are hydrophobic, electrically neutral, and spatially similar to aspartate and glutamate. Asp125, Asp152, Glu195, and Asp276 were found to be inactive, but still had the ability to bind. Asp125 and Asp152 are involved in SAM binding, and Glu195 and Glu276 residues are involved in sterol binding (34). Because the histidine side chain can exchange protons near physiological pH, it often plays a role in enzymatic catalysis involving proton transfer; therefore, histidine residues at position 90, 107, 199, and 238 were previously leucine-screened in ScSMT with only His90 being essential by becoming inactive after substitution with leucine (34). Sterol and SAM were bound to His90Leu with similar efficiency as the wild type enzyme, suggesting that His90, part of region I, acts directly in catalysis and may be responsible for the deprotonation step that generates the methylene structure.
4.5 Homology Model The role of the specific amino acid residues in crucial interactions at the active site in yeast SMT has been proven through the mutagenesis of the conserved acidic, histidine residues and Tyr81. Functional analysis of these highly conserved amino acids in the primary structure of ERG6 by site-directed mutagenesis have led to a secondary structure prediction of the enzyme (Figure 32) and a homology 3-deminsional model of Trypanosoma brucei (Tb) SMT with zymosterol constructed by collaborators (Figure 33) (7). The secondary structure model shows 10 α-helices and 6 β-sheets separated by a series of loops. The location of the α-helices and β-sheets generally correlated well with an average hydropathy plot for the set of cloned SMTs studied to date (29). The homology model of TbSMT, incorporates all of the key structural features of sterols in its mechanism of binding to the C24-SMT enzyme based on the previous binding studies with T. brucei and S. cerevisiae.
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Figure 32. The predicted secondary structure of SMT showing α-helices, β-sheets and loops. The relevant secondary structural units are numbered by their position in the alignment shown in Figure 29. The four conserved substrate binding domains are labeled I to IV with sterol acceptor and methyl donor docked in the model based on the results of several lines of evidence discussed in the text. A hydropathy plot for the S. cerevisiae SMT is shown illustrating the location of the substrate binding segments indicated in the model above (29).
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Figure 33. Ribbon representation of the active site of TbSMT. The AdoMet- (SAM; magenta) and sterol- (STE; orange) binding pockets are shown. Tyrosine residues that were substituted are labelled in blue. Relevant contact amino acids identified in the homology modelling are shown in stick representation. The image was using PyMol with coordinates obtained by homology modelling against CMA coordinates as described in Materials and methods section (23).
The sterol binding pocket incorporates α-helix turn motifs rich in aromatic amino acids proposed to be important in cation-π interactions involved with stabilization of the enzyme-bound intermediate (16). In yeast SMT, residues 81–86 form the conserved motif of aromatic residues which is of considerable importance in inhibitor binding as previously reported from our lab and in this model Tyr66 (the Tyr81 equivalent in TbSMT) is in close proximity to the sterol side chain (36). This model actually revealed a 69 Texas Tech University, Alicia L. Howard, December 2016
histidine residue of the C24-SMT to come in close contact with the C3-hydroxyl group. It would appear at position His223 there is a contact amino acid with polar residues. Histidine has two titratable –NH groups that can take the role of a nucleophile or electrophile when it’s protonated or deprotonated. There may be electrostatic interactions between the negatively charged zymosterol and the positively charged amino group of the histidine residue of TbSMT that contributes to the affinity and specificity of binding as well as to the orientation of the substrate in the binding site and structure of the complex formed. His90, part of region I, acts directly in catalysis and may be responsible for the deprotonation step that generates the methylene structure. His90 in Yeast SMT is equivalent to His75 in TbSMT and looks to be positioned close to the double bond of the side chain of the substrate. The Asp110 and Glu180 (Asp125 and Glu195 ScSMT equivalents respectively) appear to agree with the binding studies previously reported. The Asp110 is in close proximity to be involved in SAM binding while the Glu180 is close to the sterol for binding of the substrate.
The 24-alkylation process involves an SN2 nucleophilic attack from a positively charged donor (SAM) to an acceptor molecule which is electron rich at C-24 by virtue of a Δ24-double bond (37). Based on this model, there’s an Asp110 residue (equivalent to yeast SMTs Asp125) that’s in the SAM binding pocket that when replaced with a leucine residue, becomes inactive. However, it can still bind sterol. There’s a loss of 3kJ/mol in binding energy for SAM binding with the Asp110Leu mutant (34). Referring back to the yeast growth studies with abasol, the newly-made commercial drug inhibits the C24-SMT by binding in the SAM binding pocket. Its structural features are very similar in size and electronics. Abasol has an aromatic ring that mimics SAMs imidazole ring that has the potential to become protonated to carry a positive charge. The imidazole ring seems to be interacting with the acidic residue aspartate. Based off the homology model, abasol could be interacting within this SAM binding site in the same manner as well. When tested with 14 L-[methyl- C]-methionine, abasol inhibited incorporation of radioactivity into sterol at 10 ng/mL, concentrations lower than the 1 µg/ml with sinefungin, a competitive inhibitor for SAM. Not much information concerning residues essential for inhibitor recognition of this class of membrane-associated sterol catalyst has been described in depth so these 70 Texas Tech University, Alicia L. Howard, December 2016
models provide insight into what amino acids are involved with the binding of SAM and sterol (33).
4.6 Molecular Docking Molecular docking study is an efficient technique to predict the potential ligand binding site(s) on the whole protein target (36). Modeling profiles are generated from the refined domains of methyltransferase superfamily to determine which proteins align with the models (36). Sequence alignment of the SMT from L. infantum and T. brucei showed a high sequence homology suggesting the striking similarity of motifs and domains within the family (Figure 34). Multiple sequence alignments highlighting the active site residues demonstrates the protein conservation among the two species. Both the substrate binding sites and AdoMet binding domain are conserved among both organisms as well as the ATP binding site. In order to explore the characteristics of the binding pocket of the enzyme and to obtain the structural requirements for possible better inhibition activities, molecular docking was carried out by Azam and coworkers (36).
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Figure 34. Alignment of sterol C24-methyltransferase amino acid sequence (GenBank accession numbers) from L. infantum (ADX32472.1) and T. brucei (AAZ40214.1). Conserved regions are boxed in green with residues highlighted in red corresponding to Azams model and the blue highlighted residues corresponding to the TbSMT model.
This model depicts similar amino acids in the active site as in the homology model from TbSMT with differences in the position of the residues. The residues involved in the conformation of the 24(R,S),25-epiminolanosterol in the binding site of SMT enzyme in the Azams model is shown in (Figure 35). It was studied in detail in order to extract useful information about its conformation in the binding pocket of the SMT enzyme. Azam found this compound docked deeply into the active site region making interactions with the residues Gln139, Gly112, Gly114, Tyr68, Gly71, Gln72 Asn136, Asn134, Asn135, Glu180. The backbone of Ile112 is forming a hydrogen bond (–O___H–O). The presence of H-bond with the solvent-exposed pocket of the receptor (Ile112) further stabilizes the interaction. This hydrogen bonding agrees with the work of PbSMT tested with varies functional groups positioned at C3 on lanosterol by failing to bind to the ammonium salt nor the C3-desoxy. The hydroxyl group of the sterol is
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involved in hydrogen bonding with possibly the backbone of the polypeptide. Hydrophobic interactions are formed in the majority of all the ligand–protein interactions (36). Due to this high hydrophobic character of the binding pocket, it’s understood that hydrophobic substitutions tend to enhance the fitting of each mechanism based inactivator in the binding site (36). This can be explained considering that the increase of the surface complementarity forces the inhibitor to maintain a stable conformation.
Figure 35. Interactions of 24(R,S),25-epiminolanosterol in the binding site of SMT enzyme are shown in 2D conformation with surrounding residues using TbSMT amino acid numbering (36).
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The secondary and three-dimensional structures along with the scanning mutagenesis and related replacement experiments to date of 77 Erg6p amino acids, which make up roughly 20% of the Erg6p amino acids, are not consistent with one amino acid residue playing a greater role in catalytic outcome than another; rather, they support a model wherein the identified residues play a shared role in product formation (Table 9,10,11) (29). Thus, product diversity that evolves from catalytic promiscuity in the yeast SMT mutants appears to be determined by the collective action of key residues in the sterol binding segments that contact the acceptor side chain (29).
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Table 9 Catalytic competence of S. cerevisiae mutants made in the Nes lab.
Enzyme Mutant Substrate Results ScSMT E64L S87L A193S L348L Zymosterol 81-100% activity D67L S87Q T197L A350L Y74L S87E C198L G351L D79L F89L K215L S354L Y81L E98L G217L F357L Y81F V130L G218L E82L Y153L Y223L E82Q Y153F W225L Y83L F188L D229L Y83W D189L H238L Y83F Y192L S88L A196L P216L 61-80% activity I194L E209L E224L Y81L G132L P201L 36-60% activity E82D C128S V349L W85L H199Q K353L Y81I F91L H199R A221L 10-35% activity G84L H107L T216L V222L G86L E108L C198V Y278L S88N F178L F220L E68L V126L A193L D276L 0-9% activity T78V G127L E195L W286L Y81V C128L C198F G347L Y81A G129L H199L G352L H90L G131L A200L H90Q P133L Y207L H90R D152L E246L D125L F188L F269L Y81F Y192F G218L Y223F Zymosterol C2 transfer D79L G217L T219L Fecosterol activity Y74F Y192F W85F Zymosterol 51-100 C1 Y81F Y207F W225F transfer activity Y153F Y223F W286F E82L Δ25,27 Y81W 124% activity Y81F Y81W 26,27- >100% activity dehydrozymosterol Y81I Y81L 24-40% activity Y81V Y81A 15% activity Y81W Increase in C26- monol
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Table 10 Catalytic competence of fungal mutants made in the Nes lab.
Enzyme Mutant Substrate Results CrSMT Y110L Y110A Y110G Cycloartenol 1-10% activity Y110V 30% activity Y110F S360G S360Y 45-65% activity Y110W S360N S360L 0% activity Y110W Y110A S360L S360N 24(28)-Methylene 0-1% activity Y110L Y110G S360G S360Y Cycloartenol Y110V Y110F 60% activity Y110G S360L Zymosterol 0-5% activity Y110A Y110V 15% activity Y110L S360Y 40-60% activity Y110F S360N 68% activity Y110W 75% activity
Change in product ratio
S360G >100% activity S360L 24-Methylene 0% activity Lophenol S360N S360Y 20-30% activity S360G 89% activity S360L S360G Obtusifoliol 0-15% activity S360N 50% activity S360Y 83% activity TbSMT Y66F Y177F Y223F Zymosterol 23-40% Km/Vmax
No Change in C2 transfer activity Y66F Increase in ergosta- 8,25(27)-dienol and ergosta-8,24(28)-dienol
PbSMT Y88L Lanosterol 50% activity Y88F 90% activity
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Table 11 Catalytic competence of plant mutants made in the Nes lab.
AtSMT W89F W89A Y85E Cycloartenol 0% activity W89G W89L Y85A W89Y Y85W 5-10% activity Y85F Y85L 20-25% activity W89G W89L Y85A 24-Methylene 0% activity W89A Y85E Y85L Lophenol Y85W 5% activity W89F W89Y Y85F 20-30% activity GmSMT1 S81L F85L Y194F Y225F Cycloartenol 100% activity of F82L W87L G219L D278L C1 transfer F82I F91L Q220L D278Q Y83L F93L Y221L D278R Y83F Y194L Y225L E84L G341L G341N 45-65% activity G341S G341Y F82L W87L G341L G341N 24(28)-Methylene Loss of C2 Y83L F91L G341S G341Y Cycloartenol transfer activity F85L F93L S81L Y194L Y221L D278Q No change in C2 Y83L Y194F Y225L D278R transfer Y83F G219L Y225F E84L Q220L D278L
Y83F 24(28)-Methylene Preferred Lophenol substrate of CA Different product ratio with increase in 24(28)Z- Ethylidene Y194F Y221L Y225L Y225F Cyclobranol Increase in activity S81L Decrease in activity Y83F Change in Profile Diol sterols observed
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CHAPTER V DISCUSSION
Sterols are indispensable compounds found in all eukaryotes because of their structural components involved in the plasma membrane structure (3). In mammals, insects, and higher plants, sterols are the precursor to the steroidal hormones that are essential to eukaryotes’ wellbeing and play a wide range of cellular functions in pheromone signaling and in sparking cells to proliferate (13). Because of their chemical significance, the main pathway of sterol biosynthesis has been widely studied with many different types of research conducted. Phytosterol biosynthesis, characterized by the formation of sterols with a 24-alkyl group in the side chain, involves multiple intermediates and enzymatic systems arranged in photosynthetic and non-photosynthetic organism (29). It is this 24-alkyl group that separates these sterols from those found in mammals, which is catalyzed by the enzymes collectively known as sterol C24- methyltransferase that play a key role in sterol biosynthesis in different pathogenic organisms by setting the pattern of the side chain structure of the final product (15). Although absent in humans, this catalyst provides critical pathway-specific enzymatic steps by generating intermediates that ultimately are converted to end products that control plant and fungal physiology (15). Despite the significant advances that have been made, a molecular-level understanding of the C24-SMT active site and its requirements for sterol biosynthesis remains ambiguous. For decades there has been interest in unearthing the molecular libraries (metabolite structure and amounts) that characterize the sterol metabolomes involved in growth and developmental regulation in the hopes that catalyst-based sterol analogs can be designed to control flux at the rate-determining step of the pathway (13). In general, the C24-SMT is consider the rate-limiting step that governs the methylation at C24 with precision to generate single or multiple products so inhibition of these enzymes can result in depletion of cellular 24-alkyl sterols, such as ergosterol, accompanied by growth inhibition (13). Ergosterol is one of the key components of the fungal cell membrane and, as the main sterol of yeast, it is necessary for the growth of cells and normal membrane
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fluidity, asymmetry and membrane integrity, and contributes to the proper function of membrane-bound enzymes (26). The fungal ability to C-methylate sterols is the only proven difference in sterol synthetic pathways between humans and opportunistic pathogens (34). Having greater knowledge of the way sterols vary across different kingdoms and how they operate in the cell can help lead to the discovery of new therapeutic strategies to target harmful microbes and a better understanding on how genetic defects in these biosynthetic pathways can harm the human body. Interest in 24-alkyl sterol diversity and function stems from the SMT enzyme’s regulatory properties to control 24-alkyl sterol production (30). A series of enzyme inhibitors against SMT started to evolve by the end of the 1980s with the purpose to obtain insight into the mechanism of the C24-methylation reactions by gaining information on the active site topography and develop leads to the disruption of phytosterol homeostasis associated with disease states (16,36). A list of sterol analogs had been studied in vitro and in vivo to impair SMT activity irreversibly which provided leads for the design of anti-fungal inhibitors (36). To focus on the rational design and development of specific inhibitors of the 24-SMT, two transition state-based and product inhibitors were evaluated with S.cerevisiae SMT: 25-Azalanosterol, a substrate analog with a nitrogen in the side chain, and Abasol, an antifungal agent that inhibits the methylene transfer to the sterol side chain from SAM. These compounds were shown to inhibit SMT activity with IC50 values that range from 1 µM (25-azalanosterol, a transition state analog) to 7 μM (abasol, a product inhibitor), which are IC50 values that are similar to several known drugs made available that interfere with sterol biosynthesis which are used to treat diseases caused by opportunistic pathogens. Knowing the full inhibitory effects allows the two compounds to possibly be involved in combination drug therapy that will first stop cell growth followed by the death of the pathogen. The C24-SMTs have been difficult to study because they are in low abundance in wild-type organisms with no X-ray crystal structure determination, resulting in limited opportunity provided to investigate the basic chemical reactions carried out by these enzymes using classic approaches (16). Locating catalytically relevant amino acid residues and regions of the SMT was made possible from sequence alignments of SMTs 79 Texas Tech University, Alicia L. Howard, December 2016
across kingdoms and site-directed mutagenesis of select, histidines, acidic and aromatic residues considered important to the coupled methylation-deprotonation reaction that led to the determination of signature motifs throughout the primary sequence. Mutational analyses of the amino acids within these signature motifs has provided information about the size and shape of the enzyme binding pocket and the identity of amino acids involved with catalysis (15). Analysis on a series of 27 mutants across kingdoms revealed mutations that affected channeling by converting substrate to product with ratios that favored the formation of Δ25(27)-olefins over the preferred Δ24(28)-olefins normally found in the wild type SMT. There were also a few essential amino acids that loss activity that were either considered to be involved directly with catalysis or with binding of the substrate and/or SAM. The inhibitor studies that mimic the transition state of the reaction, that are involved with SAM binding, along with mutagenesis studies of conserved residues in the five regions of the primary sequences helped verify the C24-SMT homology model of Trypanosoma brucei SMT. The model incorporates many of the key structural features of the enzyme in its mechanism of binding to sterol and SAM that was based on the previous binding studies with T. brucei and S. cerevisiae. The combination of mutagenesis used to identify key amino acid residues in the active site and the inhibition profiles with ScSMT have overall provided a greater knowledge of the sterol C24- methyltransferase catalyzed mechanism.
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2. Nes, W.D. (2011) Biosynthesis of cholesterol and other sterols, Chem. Rev. 111, 6423-6451 3. Desmond, E., and Gribaldo, S. (2009) Phylogenomics of Sterol Synthesis: Insights into the Origin, Evolution, and Diversity of a Key Eukaryotic Feature, Genome. Biol. Evol. 1, 364-381
4. Perlin, D. (2007) Resistance to echinocandin-class antifungal drugs, NIH. 11, 121-130 5. Song, Z., and Nes, W.D. (2007) Sterol biosynthesis inhibitors: Potential for transition state analogs and mechanism-based inactivators targeted at sterol methyltransferase, Lipids. 42, 15-33
6. Porter, F.D., and Herman, G.E. (2011) Malformation syndromes caused by disorders of cholesterol synthesis. Journal of Lipid Research. 52, 6-34 7. Volkman, J. (2005) Sterols and other triterpenoids: source specificity and evolution of biosynthetic pathways, Organic Geochemistry. 36, 139-159
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11. Kaneshiro, E.S. (2002) Sterol biosynthesis in Pneumocystis: unique steps that define unique targets, D. Resis. Updt. 5, 259-268 12. Zhou, W., Song, Z., Kanagasabai, R., Liu, J., Jayasimha, P., Sinha, A., Veeramachanemi, P., Miller, M.B., and Nes, W.D. (2004) Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis, Molecules. 9, 185-203
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13. Pereira, M., Song, Z., Santo-Silva, L.K., Richards, M.H., Nguyen, T.T.M., Liu, J., Soares, C.M.A., Cruz, A.H.S., Ganapathy, K., and Nes, W.D. (2010) Cloning, mechanistic and functional analysis or a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis, Biochimica et Biophysica Acta. 1801, 1163-1174
14. Howard, A.L., Liu, J., Elmegeed, G.A., Collins, E.K., Ganatra, K.S., Nwogwugwu, C.A., and Nes, W.D. (2012) Sterol C24-methyltransferase: Physio- and stereo-chemical features of the sterol C3 group required for catalytic competence, Biochemistry and Biophysics. 521, 43-50
15. Nes, W.D. (2003) Enzyme mechanisms for sterol C-methylation, Phytochemistry. 64, 75-95
16. Liu, J., and Nes, W.D. (2009) Steroidal Triterpenes: Design of Substrate-Based Inhibitors of Ergosterol and Sitosterol Synthesis, Molecules. 14, 4690-4706
17. Nes, W.D., Song, Z., Dennis, A.L., Zhou, W., Nam, J., and Miller, M.B. (2003) Biosynthesis of phytosterols, Kinetic mechanism for the enzymatic C-methylation of sterols, The Journal of biological chemistry. 278, 34505-16
18. Nes, W.D., Marshall, J.A., Jia, Z., Jaradat, T.T., Song, Z., and Jayasimha, P. (2002) Active site Mapping and Substrate Channeling in the Sterol Methyltransferase Pathway, The Journal of Biological Chemistry. 8, 42549-42556
19. Kodedova, M., and Sychrova, H. (2015) Changes in the Sterol Composition of the Plasma Membrane Affect Membrane Potential, Salt Tolerance and the Activity of Multidrug Resistance Pumps in Saccharomyces cerevisiae, PLoS ONE. 10, 1-19
20. Jensen-Pergakes, K.L., Kennedy, M.A., Lees, N.D., Barbuch, R., Koegel, C., and Bard, M. (1998) Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants, Antimicro. Agents and Chemo. 45, 1160-7
21. Vandeputte, P., Tronchin, G., Berges, T., Hennequin, C., Chabasse, D., and Bouchara, J. (2007) Reduced Susceptibility to Polyenes Associated with a Missense Mutation in the ERG6 Gene in a Clinical Isolate of Candida glabrata with Pseudohyphal Growth, Antimicro. Agents and Chemo. 51, 982-990
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22. Lorente, S., Rodrigues, J., Jimenez, C., Joyce-Menekse, M., Rodrigues, C., Croft, S., Yardley, V., Luca-Fradley, K., Ruiz-Perez, L., Urbina, J., Souza, W., Pacanowska, D., and Gilbert, I. (2004) Novel Azasterols as Potenial Agents for Treatment of Leishmaniasis and Trypanosomiasis, Antimicrobial Agents and Chemotherapy. 48, 2937-2950
23. Liu, J., Ganapathy, K., Wywial, E., Bujnicki, J., Nwogwugwu, C.A., and Nes, W.D. (2011) Effect of substrate features and mutagenesis of active site tyrosine residues on the reaction course catalyzed by Trypanosoma brucei sterol C-24- methyltransferase, Biochem. J. 439, 413-422
24. Souza, W., and Rodrigues, J.C.F. (2009) Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs, Interdisciplinary Perspectives on Infectious Diseases. 2009, 1-19
25. Nes, W.D. (2000) Sterol methy transferase: enzymology and inhibition, Molecular and Cell Biology of Lipids. 1529, 63-88
26. Borelli, C., Schaller, M., Niewerth, M., Nocker, K., Baasner, B., Berg, D., Tiemann, R., Tietjen, K., Fugmann, B., Lang-Fugmann, S., and Korting, H.C. (2008) Modes of Action of the New Arylguanidine Abafungin beyond Interference with Ergosterol Biosynthesis and in vitro Activity against Medically Important Fungi, Chemotherapy. 54, 245-259
27. Marshall, J. (2001) Studies on the enzymology of sterol methyltransferase from Saccharomyces cerevisiae, Tex Tech Univ, PhD Diss. 1-95
28. Ramakrishnan, C., Dani, V.S., and Ramasarma, T. (2002) A conformational analysis of Walker motif A GXXXXGKT (S) in nucleotide-binding and other proteins, Protein engineering. 15, 783-98
29. Ganapathy, K., Jones, C., Stephens, C., Vatsyayan, R., Marshall, J., and Nes, W.D. (2008) Molecular probing of Saccharomyces cerevisiae sterol 24-C methyltransferase reveals multiple amino acid residues involved with C2-transfer activity, Biochim. Biophy. Acta. 1781, 344-351
30. Nes, W.D., McCourt, B.S., Marshall, J.A., Ma, J., Dennis, A.L., Lopez, M., Li, H., and He, L. (1999) Siter-Directed Mutagenesis of the Sterol Methyl Transferase Active Site from Saccharomyces cerevisiae Results in Formation of Novel 24-Ethyl Sterols, J. Org. Chem. 64, 1535-1542
31. Dalby, P. (2011) Strategy and success for the directed evolution of enzymes, Curr Opin Chem Biol. 21, 473-480. 83 Texas Tech University, Alicia L. Howard, December 2016
32. Holde, K.E., and Mathews, C.K. (1995) Biochemistry -2nd ed, Benjamin/Cummings Publishing Company, Inc, Menlo Park.
33. Nes, W.D., Jayasimha, P., and Song, Z. (2008) Yeast sterol C24- methyltransferase: Role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis, Archives of Biochemistry and Biophysics. 477, 313-323
34. Nes, W.D., Jayasimha, P., Zhou, W., Kanagasabai, R., Jin, C., Jaradar, T.T., Shaw, R.W., and Bujnicki, J.M. (2004) Biochemistry. 43, 569-576
35. Haubrich, B., Howard, A., Adams, L., Jayasimha, P., Wang, Q., Snell, W., Collins, E., Miller, M., Thomas, C., Pleasant, S., and Nes, W.D. (2013) Characterization, mutagenesis and mechanistic analysis of an ancient algal sterol C24-methyltransferase: Implications for understanding sterol evolution in the green lineage, Phytochemistry. 113, 64-72
36. Azam, S.S., Abro, A., Raza, S., and Saroosh, A. (2014) Structure and dynamics studies of sterol 24-C-methyltransferase with mechanism based inactivators for the disruption of ergosterol biosynthesis, Mol. Biol. Rep. 41, 4279-4293
37. Nes, W.D., Janssen, G.G., and Bergenstrahle, A. (1991) Structural Requirements 24(25) for Transformation of Substrate by the (S)-Adenosyl-L-methionine:Δ -Sterol Methyl Transferase, The Journal of Biological Chemistry. 266, 15202-15212
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APPENDIX A MASS SPECTRA OF STUDY STEROLS
A)
B)
Figure 36. Mass Spectrometer for the sterol composition found in ScSMT wild type growth studies; panel A) Zymosterol; panel B) Ergosterol; panel C) Fecosterol and panel D) Δ7-Fecosterol.
85 Texas Tech University, Alicia L. Howard, December 2016
C)
D)
Figure 36. Continued. Mass Spectrometer for the sterol composition found in ScSMT wild type growth studies; panel A) Zymosterol; panel B) Ergosterol; panel C) Fecosterol panel and D) Δ7-Fecosterol.
86 Texas Tech University, Alicia L. Howard, December 2016
A)
B)
Figure 37. Mass Spectrometer for the sterol composition found in ScSMT wild type growth studies when incubated with inhibitors; panel A) Zymosterol; panel B) Δ5,7,24- Cholestatrienol; panel C) Δ7-Zymosterol and panel D) Δ5,7,22,24-Cholestatetraenol
87 Texas Tech University, Alicia L. Howard, December 2016
C)
D)
Figure 37. Continued. Mass Spectrometer for the sterol composition found in ScSMT wild type growth studies when incubated with inhibitors; panel A) Zymosterol; panel B) Δ5,7,24-Cholestatrienol; panel C) Δ7-Zymosterol and panel D) Δ5,7,22,24-Cholestatetraenol
88 Texas Tech University, Alicia L. Howard, December 2016
APPENDIX B OLIGONUCLEOTIDES SEQUENCES
Table 12 DNA sequence of the synthetic oligonucleotides used for site-directed mutagenesis of the ERG6 cDNA
Mutant Oligonucleotide 5’ Tm S. cerevisiae G347L CTA GAA AAT GCT GCG GTT CTT TTA GTT GCC GGT GGT AAG 64.9°C G347V CTA GAA AAT GCT GCG GTT GTT TTA GTT GCC GGT GGT AAG 65.1°C G347A CTA GAA AAT GCT GCG GTT GCT TTA GTT GCC GGT GGT AAG 66.1°C G347N CTA GAA AAT GCT GCG GTT AAT TTA GTT GCC GGT GGT AAG 63.8°C G347Y CTA GAA AAT GCT GCG GTT TAT TTA GTT GCC GGT GGT AAG 63.8°C G347S CTA GAA AAT GCT GCG GTT AGT TTA GTT GCC GGT GGT AAG 64.6°C V349L GCT GCG GTT GGT TTA CTT GCC GGT GGT AAG TCC 67.7°C G352L GGT TTA GTT GCC GGT CTT AAG TCC AAG TTA TTC 60.3°C C198F GCA ATT GAG GCC ACA TTT CAC GCT CCA AAA TTA GAA 63.5°C C. reinhardtii S360L GTG GAG GTT GCC AAG CTG CTC ATC CAG GGC GGC 72.1 °C S360G GTG GAG GTT GCC AAG GGG CTC ATC CAG GGC GGC 73.3 °C S360Y GTG GAG GTT GCC AAG TAC CTC ATC CAG GGC GGC 70.4 °C S360N GTG GAG GTT GCC AAG AAC CTC ATC CAG GGC GGC 70.1 °C Y110F CTA GTC ACT GAC ATT TTC GAG TGG GGC TGG GGC 67.0°C Y110W CTA GTC ACT GAC ATT TGG GAG TGG GGC TGG GGC 68.4°C Y110L CTA GTC ACT GAC ATT CTC GAG TGG GGC TGG GGC 67.9°C Y110V CTA GTC ACT GAC ATT GTC GAG TGG GGC TGG GGC 68.1°C Y110A CTA GTC ACT GAC ATT GCC GAG TGG GGC TGG GGC 69.8°C Y110G CTA GTC ACT GAC ATT GGC GAG TGG GGC TGG GGC 69.8°C G. max SMT1 G341L GAG AAG GCT GCA GAG CTA CTA GTT GAA GGA GGG 64.8 °C G341S GAG AAG GCT GCA GAG TCT CTA GTT GAA GGA GGG 64.6 °C G341Y GAG AAG GCT GCA GAG TAC CTA GTT GAA GGA GGG 64.7 °C G341N GAG AAG GCT GCA GAG AAC CTA GTT GAA GGA GGG 65.0 °C
The mutagenic codons are highlighted in red
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