ABSTRACT EXAMINATION of MITOCHONDRIAL CIT3 in SACCHAROMYCES CEREVISIAE AS the GENE for METHYLCITRATE SYNTHASE by Eric R. Graybil

ABSTRACT

EXAMINATION OF MITOCHONDRIAL CIT3 IN SACCHAROMYCES CEREVISIAE AS THE GENE FOR METHYLCITRATE SYNTHASE

By Eric R. Graybill

The existence of a methylcitrate pathway for propionate metabolism in yeasts has been shown but the 2-methylcitrate synthase in this pathway had not been clearly identified. In this study, the product of the CIT3 gene has been identified as a dual specific citrate and methylcitrate synthase and that of the CIT1 gene as a specific citrate synthase. Cit1p 3 -1 -1 has catalytic activity only with acetyl-CoA as substrate (kcat / Km = 150x10 M s ) 3 whereas Cit3p has similar catalytic efficiencies with both acetyl-CoA (kcat / Km = 9x10 -1 -1 3 -1 -1 M s ) and propionyl-CoA (kcat / Km = 10x10 M s ). Deletion of CIT1 produces a shift in the ratio of these two activities towards greater methylcitrate synthase activity. 13C-NMR spectroscopic analysis of pyruvate metabolism revealed an accumulation of acetate and isobutanol in Δcit3 but not in wild-type or Δcit1. These data indicate that Cit3p is the methylcitrate synthase in Saccharomyces cerevisiae.

EXAMINATION OF MITOCHONDRIAL CIT3 IN SACCHAROMYCES CEREVISIAE AS THE GENE FOR METHYLCITRATE SYNTHASE

A Thesis

Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Masters of Science Department of Chemistry and Biochemistry by Eric R. Graybill Miami University Oxford, Ohio 2006

Advisor Dr. John W. Hawes

Reader Dr. Michael W. Crowder

Reader Dr. Ann E. Hagerman

Reader Dr. Gary A. Lorigan

Table of Contents

1. Introduction: Saccharomyces cerevisiae 1 1.1 Yeast as a Model Organism 1 1.2 Relevance of Propionate Metabolism 1 1.3 Disease States Associated with Propionate Metabolism 2 1.4 Propionate Metabolism in Salmonella typhimurium 2 1.5 2-Methylcitric Acid Cycle in Yarrowia lipolytica 3 1.6 Evidence for 2-Methycitric Acid Cycle in 4 Saccharomyces cerevisiae 1.7 Enzymes of the 2-Methylcitric Acid Cycle in 4 Saccharomyces cerevisiae 1.8 Citrate Synthases in Saccharomyces cerevisiae 5 1.9 Hypothesis and Goals 6 1.10 References 11 2. Characterization of Mitochondrial CIT1 and CIT3 to Determine 13 the Methylcitrate Synthase Gene 2.1 Introduction 13 2.2 Materials and Methods 14 2.2.1 Materials 14 2.2.2 Methods 15 2.2.2.1 Comparison of amino acid sequences. 15 2.2.2.2 Cloning of CIT1 and CIT3 cDNAs. 16 2.2.2.3 Cit1p over-expression and purification. 16 2.2.2.4 Cit3p over-expression and purification. 17 2.2.2.5 Coenzyme A Thio-ester synthesis. 17 2.2.2.6 Enzyme assay conditions for measurement of 18 citrate and methylcitrate synthase activities. 2.2.2.7 Wild-type, Δcit1, Δcit3 sample preparation for 18 native enzyme activity analysis. 2.2.2.8 Preparation of yeast samples for 13C-NMR 19

analysis. 2.2.2.9 Analysis by 13C-NMR. 19 2.2.2.10 GC-MS examination of isobutanol in wild-type 20 and Δcit3. 2.3 Results and Discussion 20 2.3.1 Structural sequence comparison. 20 2.3.2 Cloning of CIT1 and CIT3 cDNAs. 21 2.3.3 Expression and purification of Cit1p and Cit3p. 21 2.3.4 Substrate specificities of Cit1p and Cit3p. 22 2.3.5 Effect of gene knockouts on native citrate and 23 methylcitrate synthase activities. 2.3.6 Propionate metabolism in wild-type and mutant strains. 24 2.3.7 Pyruvate metabolism in wild-type and mutant strains. 24 2.3.8 Analysis of isobutanol by GC-MS. 26 2.4 Conclusions 26 2.5 References 40

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List of Tables

2-1: List of primers used to generate CIT1 and CIT3 cDNAs. 36 2-2: Percent identity and similarity among citrate synthase homologues. 37 2-3: Substrate specificities of recombinant Cit1p and Cit3p. 38 2-4: Native citrate synthase and methylcitrate synthase activities of 39 WT, Δcit1, and Δcit3.

List of Figures 1-1: Methylmalonyl-CoA pathway. 7 1-2: 2-Methylcitric acid pathway. 8 1-3: Comparison of TCA cycle and methylcitric acid pathway. 9 1-4: Model of propionate metabolism via the 2-methylcitric acid pathway in 10 Saccharomyces cerevisiae. 2-1: Amino acid sequence comparison of citrate synthase and a 28 methylcitrate synthase homologues. 2-2: SDS PAGE gel electrophoretic analysis of recombinant Cit1p 30 and Cit3p. 2-3: 13C-NMR spectroscopy of wild-type S. cerevisiae in YPD with 31 2-13C-propionate. 2-4: 13C-NMR spectroscopy of S. cerevisiae grown in YPD with 32 2-13C pyruvate. 2-5: Model of pyruvate flux in wild-type and mutant 33 Saccharomyces cerevisiae. 2-6: 13C-NMR spectroscopy of Δpda1 S. cerevisiae grown in YPD with 34 2-13C pyruvate. 2-7: GC-MS analysis of standard isobutanol and the Δcit3 strain. 35

Abbreviations

Chapter 1 coenzyme A (CoA) open reading frame (ORFs) Saccharomyces Genome Database (SGD) tricarboxylic acid (TCA)

Chapter 2 dithiobis(2-nitrobenzoic acid) (DTNB) Isopropyl-β-D-thiogalactopyranoside (IPTG) phosphate-buffered saline (PBS) pyruvate dehydrogenase (PDH) tryptic soy broth (TSB) wild-type (WT) yeast, tryptone, dextrose (YPD) yeast, tryptone, ethanol (YPE) yeast, tyrptone, glycerol (YPG) In this thesis we have followed conventional nomenclature for designation of yeast genes, proteins, and mutants. Gene names are given in uppercase italic lettering (example CIT1). Protein is referred to with the gene name followed by the letter p (example Cit1p). Mutant strains are designated with a Greek letter delta followed by the gene name in lowercase italic letters (example Δcit1).

Acknowledgements I would like to thank all of my committee members for allowing me to travel the path that I wanted to follow no matter how difficult I wanted to make it on myself. I would like to thank Miami University for providing free access to places such as the recreational center (of which I did not take as much advantage as I should have) and sporting events such as football and hockey. Both opportunities allowed me to experience moments I will cherish for a long time. I would like to thank Dr. Ian Peat for great support and freedom when I was the MALDI teaching assistant and also for showing that documentation of procedure well written and in simple terms is invaluable. I would also like to thank the different members of my lab group starting with my advisor John Hawes for showing me that a good ole’ Indiana boy could be quite successful in the world of biochemistry research. Kerry Lucas, who showed that if I had worked the way I envisioned from the start that success would have come early and often. Chris Chailland, who I argued with like we were two old grizzled politicians dead set in our ways and showed me that at the end of the day all that mattered were results in the lab. Matt Rouhier, my hero, who in a short time demonstrated that a good person could indeed make it and be successful in this very tough world of research. I would also like to thank all of the members of the graduate class that initially started with me in August 2003. I had the privilege of starting with people that have the unique ability of being highly talented, highly motivated, very kind, and very caring. I would like to think that this has helped me to be a better person on a number of levels. Of the members of this class, I especially would like to thank David Collins, Tracy (Petersen) Mattox, Mr. Ma, and Sachin Kumar who on many occasions invited me into their homes or an event out together, allowed me to share a small part of an experience with them, and ultimately led me to having a totally unique collegiate experience as a graduate student. Additional thanks to Mat Mattox for one night naming the perception of who I was that amazingly stuck with most members of the department. Also, to Allen Easton, whose simple words during MALDI training allowed me to gather a reputation quicker than I thought would happen.

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Also a general thanks to those that I have left out either by accident or because there are too numerous of you to mention. Yet, if I did not mention Meghan Holdorf my conscience could not rest comfortably. Meghan’s one additional year of experience was invaluable in terms of all the steps that graduate school required, in addition whenever I needed a supply that our lab did not have she helped no matter how annoying I became, and also in help provided to proofread my thesis. Finally I would especially like to thank my parents who at first and many times after, did not believe me when I explained to them the financial situation that graduate school was going to be. Graduate school was my first time really living on my own and there were so many times that I called to ask questions that I knew the answer to but they still offered their advice as though it was the most important question they were asked that day. Together in almost polar opposite ways they have shaped me into the person I am today and if I look closely I see a melting pot of both of them in me.

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1. Introduction: Saccharomyces cerevisiae 1.1 Yeast as a Model Organism We examined propionate metabolism in the yeast Saccharomyces cerevisiae. Two of the most common model organisms used in the laboratory to study molecular and cellular biology are Escherichia coli, a prokaryote, and S. cerevisiae, a eukaryote. S. cerevisiae is a unicellular microbe with many genes that have homologues in humans and is therefore a good choice to study aspects of biology shared by more-complex life forms (1). S. cerevisiae has been classified as a “generally regarded as safe” organism because of its long historical use in ethanol production and as Baker’s yeast which makes it attractive to use in metabolic engineering applications (2). Metabolic engineering includes preliminary studies of mRNA, protein, and metabolite levels to determine what recombinant elements can be modified to improve desired production levels (2). The completion of the sequencing of the S. cerevisiae genome in April 1996 opened new paths for biological research including examination of open reading frames (ORFs) corresponding to known protein sequences that resulted in some predictions of S. cerevisiae protein structure and function relationships (3). Funding from the US National Institutes of Health via the National Human Genome Research Institute maintains a database of the molecular biology and genetics of S. cerevisiae, the Saccharomyces Genome Database (SGD), found on the web at http://www.yeastgenome.org/, housed at the School of Medicine, Stanford University. In addition, Open Biosystems located on the web at http://www.openbiosystems.com/ offers the ability to purchase knock-outs of all possible S. cerevisiae genes making for relatively easy and quick experiments on the gene(s) of interest. 1.2 Relevance of Propionate Metabolism Propionate is naturally found in our food and added at high concentrations as a food preservative to prevent growth of both bacteria and fungi but does not inhibit growth of yeast (4). Cellular sources of propionate include oxidation of odd chain fatty acids and breakdown of the amino acids isoleucine, valine, threonine, and methionine (5). All though propionate is a three-carbon short chain fatty acid there is a different metabolic route in different organisms. The pathway for plants is not known while yeast and bacteria use a pathway different from mammals. In mammals propionate is converted

using the methylmalonyl-CoA pathway to succinyl-CoA, which can then enter the tricarboxylic acid (TCA) cycle (Figure 1-1) (5). In the methylmalonyl-CoA pathway, propionyl-CoA first is converted by propionyl-CoA carboxylase to (S)-methylmalonyl- CoA, which is then rearranged by methylmalonyl-CoA racemase to (R)-methylmalonyl- CoA and then converted to succinlyl-CoA by methylmalonyl-CoA mutase (5). 1.3 Disease States Associated with Propionate Metabolism In humans, two well known conditions arise when propionate cannot be metabolized efficiently (6,7). These result from two autosomal recessive disorders, propionic acidemia or methylmalonic acidurias. Propionic acidemia is caused by a mutation in either the alpha or beta subunit of the mitochondrial enzyme propionyl-CoA carboxylase leading to complications in propionate utilization (6). Treatment involves dietary limitation of the amino acids that produce propionate, increase of caloric intake, avoidance of long fasting periods, and carnitine supplementation (6). Consequences include early death, and survivors may face nutritional problems with developmental delay and neurological impairment (6). A second disease in humans associated with problems of propionate utilization is methylmalonic acidurias, which involves a mutation in methylmalonyl-CoA mutase or defects in the biosynthesis of the cofactor, 5’- deoxyadenosylcobalamin (7). Conditions such as having other diseases at the same time or prolonged fasting complicate the disease leading to possible multiple organ failure and even death (7). Even with treatment patients have psychomotor retardation, pancreatitis, renal failure, and neurological deficits (7). Both of these diseases are associated with accumulation of metabolic acids from the disruption of propionate metabolism. 1.4 Propionate Metabolism in Salmonella typhimurium Salmonella typhimurium has been conclusively shown to metabolize propionate via the 2-methylcitric acid cycle, a reaction path very similar to the first half of the TCA cycle as shown in Figure 1-2 and is encoded on the prpRBCDE operon (8). Propionic acid is first converted to propionyl-CoA using propionyl-CoA synthetase, PrpE, and then condensed with oxaloacetic acid to make 2-methylcitric acid by 2-methylcitrate synthase, PrpC (9). The conversion of 2-methylcitric acid has been shown first to utilize 2- methylcitrate dehydratase, PrpD, to convert 2-methylcitric acid to 2-methyl-cis-aconitic acid, and then to 2-methylisocitric acid with an aconitase enzyme either, AcnA or AcnB

(9). 2-Methylisocitiric acid is cleaved to pyruvic acid and succinate by 2-metylisocitrate lyase, PrpB (9). Horswill et al. proposed that 2-methylcitrate or a derivative of 2- methylcitrate is the toxic compound that is responsible for bacterial growth inhibition by propionic acid (4). Yet, organisms that can metabolize propionate use 2-methylcitrate as a signaling molecule and detoxify the compound via the 2-methycitric acid cycle (4). 1.5 2-Methylcitric Acid Cycle in Yarrowia lipolytica The existence of a 2-methylcitric acid cycle was first observed in Yarrowia lipolytica. Candida lipolytica was first isolated from cheese and sausage in the 1940s, reclassified as Y. lipolytica in 1980 by van der Walt and von Arx based on aspects of its biology, and in general is a good model organism to study protein secretion (10). Tabuchi and Serizawa studied Y. lipolytica and a mutant of Y. lipolytica designated No. R-2 (11). The mutant strain showed an increase in the amount of extracellular 2- methylisocitrate when grown on odd chain alkanes and also produced small amounts of methylcitrate and methyl-cis-aconitate that correlated to the amount of available propionyl-CoA (11). Supported by their data and additional data in the literature, they proposed the methylcitric acid cycle based on the conversion of propionate by reactions 14 similar to the TCA cycle (Figure 1-3) (11). They noted that the pattern of CO2 evolution from labeled propionate was compatible with the proposed methylcitric acid cycle (11). Tabuchi and Uchiyama examined key enzymatic reactions involved in the methylcitric acid cycle in both wild-type Y. lipolytica and mutant No. R-2 (12). They showed that activity of acetyl-CoA condensing with oxaloacetate was the same between wild-type and mutant No. R-2 while activity of propionyl-CoA condensing with oxaloacetate was higher in the mutant (12). They also confirmed the enzymatic product of the condensation between propionyl-CoA and oxaloacetate as methylcitrate using paper and gas chromatography (12). The rates of isomerization with isocitrate or methylisocitrate (see Figure 1-3) were the same for wild-type and the mutant indicating that aconitase more than likely acts on both (12). The cleavage activity that converts methylisocitrate to pyruvate and succinate was 18-fold lower in the mutant. In contrast the mutant compared to the wild-type had 3-fold higher activity using isocitrate, and from these data, they proposed that the isocitrate lyase active in the TCA cycle does not cleave

methylisocitrate (12). All of the key enzymes in the 2-methylcitric acid cycle were found to be exclusively in the mitochondria except a dual location of 2-methylisocitrate lyase in both the cytoplasm and mitochondria (13). 1.6 Evidence for 2-Methycitric Acid Cycle in Saccharomyces cerevisiae Y. lipolytica is an alkane using yeast, very distantly related to other yeasts such as S. cerevisiae, and has a number of common properties with filamentous fungi (14). The main metabolic difference between Y. lipolytica and S. cerevisiae is that Y. lipolytica is an obligatory respiratory organism that catabolizes all carbons via the TCA cycle while S. cerevisiae catabolizes carbons using a variety of different methods though mainly by fermentation (15). Sumegi et al. provided evidence for the existence of the metabolon hypothesis in S. cerevisiae using labeled propionate, 13C-NMR, and the assumption that propionate metabolism followed the methylmalonyl-CoA pathway (16). However, Pronk et al. compared enzyme activities in the methylmalonyl-CoA pathway and the 2- methylcitric acid cycle and showed propionate was metabolized by the 2-methylcitric acid cycle (17). Propionyl-CoA carboxylase, which catalyzes the first step of the methylmalonyl-CoA pathway, showed an activity 100-fold lower than the consumption rate of propionate in the cultures (17). In contrast, 2-methylcitrate synthase and citrate synthase activities increased with increasing propionate-to-glucose ratios (17). Pronk et al. noted that the prior 13C-NMR analysis labeling pattern observed by Sumegi et al. would yield the same results if propionate is metabolized via the 2-methylcitric acid cycle (17). 1.7 Enzymes of the 2-Methylcitric Acid Cycle in Saccharomyces cerevisiae Complete sequencing of the S. cerevisiae genome has led to a number of ORFs with unknown functions (18). Some of these ORFs exhibit a strong homology with currently characterized yeast genes and in some cases can even restore growth if over- expressed when the other homologous yeast gene is knocked out (19). Work on different enzyme isoforms has started to identify genes that encode enzymes in the 2-methylcitric acid cycle. S. cerevisiae is known to encode two homologous genes, ACS1 and ACS2, that both code for active acetyl-CoA synthetases (20). A study of Acs1p and Acs2p showed that Acs1p could use propionate as a substrate and Acs2p could not (20), revealing Acs1p as a dual specific acetyl-CoA and propionyl-CoA synthetase. S.

cerevisiae is known to encode two highly homologous isocitrate lyases, ICL1 and ICL2, yet ICL2 does not encode a functional isocitrate lyase (19). Luttik et al. proposed that ICL2 was a 2-methylisocitrate lyase involved in propionate metabolism via the 2- methylcitric acid cycle (19). A wild-type strain was shown to have activity with 2- methyisocitrate and isocitrate while a Δicl1 and Δicl2 double mutant strain showed no activity with 2-methylisocitrate or isocitrate. Δicl2 was shown to have activity with 2- methylisocitrate and isocitrate, and a Δicl1 was shown to have activity with 2- methylisocitrate but no activity with isocitrate. These data would support that ICL2 has activity only with 2-methylisocitrate while ICL1 has activity with both isocitrate and 2- methylioscitrate (19). The localization of the 2-methylisocitrate lyase activity was shown to be in the mitochondria by use of density gradient centrifugation and use of a green fluorescent protein tag (19). Work by Epstein et al. using DNA microarray showed an increase in expression of PDH1, CIT3, ACO1, and ICL2 when propionate was added (21). They speculated that CIT3, ACO1, and ICL2 could catalyze the subsequent steps in propionate metabolism via the 2-methylcitric acid pathway of condensation, isomerization, and cleavage, respectively (Figure 1-3) (21). However, this hypothesis was based only on gene expression profiles and not on functional analyses of the proteins. 1.8 Citrate Synthases in Saccharomyces cerevisiae Although enzymes in the methylcitric acid cycle in S. cerevisiae are known, methylcitrate synthase had yet to be fully characterized (Figure 1-4) (20,21). There are 3 highly homologous citrate synthase genes in S. cerevisiae: CIT1 encoded on chromosome XIV, CIT2 encoded on chromosome III, and CIT3 encoded on chromosome XVI (22). The Cit1p has a mitochondrial leader sequence and binds to the inner surface of the inner mitochondrial membrane (23). The Cit2p has a SKL C-terminal signal sequence that targets it to the peroxisome and has been shown to participate in the glyoxylate cycle allowing yeast to use ethanol and other two-carbon compounds as sole carbon sources (24). Disruption of CIT1 results in cells that are unable to grow on acetate as a sole carbon source, and simultaneous disruption of both CIT1 and CIT2 results in glutamate auxotrophy, which is an indication of a completely citrate synthase deficient phenotype (23,25). Cit3p like Cit1p is an active citrate synthase, localized to the mitochondria, and transport of the protein into the mitochondria is dependent on the membrane potential

(22). Expression of CIT3 and CIT2 mRNAs were shown to be increased when CIT1 was deleted (22). Yet under normal conditions, the CIT3 transcript was found to be five times less abundant then the CIT1 transcript (22). Pair–wise sequence alignment of all three showed that the three conserved residues known to be important for citrate synthase activity are present (22). The regulation of CIT1 and CIT2 has been shown to be complex (22). The metabolic function of Cit3p prior to this study was unknown (23). As described above, evidence for CIT3 exclusively encoding for a methylcitrate synthase can be found in the DNA microarray work of Epstein et al. because their data showed an induction of only CIT3 and not CIT1 or CIT2 when S. cerevisiae was exposed to propionate (21). 1.9 Hypothesis and Goals We proposed that the CIT3 gene in Saccharomyces cerevisiae is the methylcitrate synthase gene. Figure 1-4 shows the methylcitric acid pathway in S. cerevisiae and enzymes that are currently known (19-21). It is quite possible that either CIT1 or CIT3 could be the methylcitrate synthase since both are located in the mitochondria and so both were examined in this study. CIT2 is peroxisomal and was not examined in this study. We compared the amino acid sequence homology to other mammalian citrate synthases and to citrate synthase and methylcitrate synthase from E. coli. We produced recombinant Cit1p and Cit3p, tested them for citrate and methylcitrate synthase activities and compared their kinetic parameters with acetyl-CoA and propionyl-CoA as substrates. We examined native citrate and methylcitrate synthase activities in wild-type and mutant strains lacking either CIT1 or CIT3 under different growth media conditions. We also examined the metabolism of 2-13C propionate and 2-13C pyruvate using 13C-NMR spectroscopy in a similar method as Escalante-Semerena and Horswill used for Salmonella typhimurium (8). These experiments were chosen to determine whether CIT1 or CIT3 is a specific methylcitrate synthase gene.

O O H

- H3CCH2 C SCoA OOC CCSCoA

Propionyl-CoA CH3

(S)-Methylmalonyl-CoA

O O H

H3CCC SCoA H2C CH2 C SCoA

COO- COO-

(R)-Methylmalonyl-CoA Succinyl-CoA

Figure 1-1: Methylmalonyl-CoA pathway. Propionyl-CoA generated by mammals is converted to succinyl-CoA.

O prpE prpC

13 13 H3C H2 CCOOH H3C H2 CCSCoA propionate propionyl-CoA

CH OH CH 3 prpD 3 acnA or acnB

13 13 HOOC CH C CH2 COOH HOOC C C CH2 COOH

COOH COOH 2-methylcitrate 2-methyl-cis-aconitate

CH O 3 prpB H H2 H2 13 13 HOOC C C CH2 COOH H3C CCOOH+ HOOC C C COOH pyruvate succinate OH COOH 2-methylisocitrate

Figure 1-2: 2-Methylcitric acid pathway. Pathway of propionate metabolism that was conclusively shown in Salmonella typhimurium (8). Included are the gene names for the enzymes known to catalyze each reaction. The 13C labeled carbon was used to differentiate between possible pathways of propionate metabolism in Salmonella typhimurium (8).

TCA Cycle Methylcitrate Cycle

acetyl-CoA Propionyl-CoA

oxaloacetate Condensation oxaloacetate

citrate 2-methylcitrate

Isomerization

isocitrate 2-methylisocitrate

Oxidative Cleavage Decarboxylation

α-ketoglutarate pyruvate + succinate

Figure 1-3: Comparison of TCA cycle and methylcitric acid pathway. These two metabolic pathways share certain reaction types prior to the last step shown, oxidative decarboxylation of isocitrate vs. cleavage of methylisocitrate.

O ACS1 CIT3 or CIT1

13 13 H3C H2 C COOH H3C H2 CCSCoA propionate propionyl-CoA

CH3 OH ACO1 CH3 ACO1

13 13 HOOC CH C CH2 COOH HOOC C C CH2 COOH

COOH COOH 2-methylcitrate 2-methyl-cis-aconitate

CH O 3 ICL2 H H2 H2 13 13 HOOC C C CH2 COOH H3C C COOH + HOOC C C COOH pyruvate succinate OH COOH 2-methylisocitrate

Figure 1-4: Model of propionate metabolism via the 2-methylcitric acid pathway in Saccharomyces cerevisiae. Prior to this study it was not known whether CIT1, CIT3, or both encode the protein that is responsible for catalyzing the reaction of propionyl-CoA and oxaloacetate to form 2-methylcitrate. Work by Epstein et al. using DNA microarray suggested that CIT3, ACO1, and ICL2 could catalyze the steps in propionate metabolism via the 2-methylcitric acid pathway (21).

1.10 References

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2. Characterization of Mitochondrial CIT1 and CIT3 to Determine the Methylcitrate Synthase Gene 2.1 Introduction This thesis describes an investigation of the mitochondrial citrate synthase genes in S. cerevisiae, CIT1 and CIT3, and the examination of their potential roles as either citrate and/or methylcitrate synthases. S. cerevisiae was used because it is a common model organism to study biology of higher eukaryotes and because of available resources such as Open Biosystems allowed purchase of gene knock-outs (1). Propionate is most commonly found as propionyl-CoA in eukaryotes and is produced by the oxidation of odd chain fatty acids and the breakdown of the amino acids isoleucine, valine, threonine, and methionine (2). While propionyl-CoA in mammalian systems is metabolized to succinyl-CoA using the methylmalonyl-CoA pathway, the pathway used in S. cerevisiae has been shown to be the methylcitric acid pathway (3). Prior work has identified genes that encode for enzymes used in the methylcitric acid cycle, ACS1 and ICL2 or ICL1 (4,5). The identity of the methylcitrate synthase gene used in the condensation of propionyl-CoA with oxaloacetate prior to this work was unknown in S. cerevisiae. The two potential candidates were CIT1 or CIT3 with both being known citrate synthases and being localized to the mitochondria (6). We proposed that the CIT3 gene in S. cerevisiae encodes for methylcitrate synthase. One aspect of this study was focused on characterizing the Cit1p and Cit3p proteins. This goal was accomplished by comparing amino acid sequences of known citrate synthases and a methylcitrate synthase. Sequence comparison using multiple alignments can supply information by identifying patterns of conserved residues to learn about function or structure of the protein (7). Some of the first work that was done after the complete sequence of the S. cerevisiae genome was obtained was to compare sequence alignments of known genes and to predict protein structure and function from the comparison (8). Prior work has already determined that the residues necessary for citrate synthase activity are conserved in all CIT genes in S. cerevisiae but work prior to this study did not examine homology to the methylcitrate synthase in E. coli (6). We also determined kinetic parameters for citrate synthase and methylcitrate synthase activities of Cit1p and Cit3p. Cit1p and Cit3p were individually cloned from

yeast DNA into a pTrcHis TOPO vector and then over-expressed and purified. The

determination of the kinetic constants Km, kcat, and kcat / Km using both acetyl-CoA and propionyl-CoA would allow us to characterize Cit1p or Cit3p as either a citrate synthase, a methylcitrate synthase, or an enzyme with dual specificity. The second focus of this study examined the genes CIT1 and CIT3 by using knock-out strains to examine activities of native enzymes, to identify metabolites using NMR, and to confirm metabolites by using GC-MS. We determined the specific activity of native citrate and methylcitrate synthase in Δcit1 and Δcit3 to determine the functional effects these gene mutations have under different media conditions. Evans et al. in 1993 used either 2-13C propionate or 3-13C propionate to study propionate metabolism in E. coli using NMR spectroscopy and observed different labeling of glutamate in correlation with the different labeled carbon of propionate (9). London et al. in 1999 used 1,2-13C labeled propionate to study propionate metabolism in E. coli using NMR spectroscopy and observed labeling of different metabolic intermediates such as trehalose and glutamate (10). Horswill and Escalante-Semerena in 1999 used 2-13C propionate to study propionate metabolism in S. typhimurium using NMR spectroscopy and observed 2- methylcitric acid cycle intermediates (11). Sumegi et al. used 3-13C propionate to study propionate metabolism in S. cerevisiae using NMR spectroscopy and observed alanine and succinate (12). Dickinson et al. has successfully used NMR to show that metabolism of valine to isobutyl alcohol uses pyruvate decarboxylase, while leucine uses a different decarboxylase to produce iso-amyl alcohol in S. cerevisiae (13,14). We confirmed the presence of some of the metabolites found in our NMR spectrum by using GC-MS. GC- MS was an ideal approach to use because 1) our metabolite of interest was easily volatilized, 2) this method allowed us to quantify, 3) the fragmentation pattern was not complex, and 4) a similar study using S. cerevisiae had been published (13,15-17). It was only by studying the protein Cit3p and CIT3 that we were able to prove our hypothesis. 2.2 Materials and Methods 2.2.1 Materials Saccharomyces cerevisiae strains BY4743 (wild-type (WT), YNR001C (Δcit1), YPR001W (Δcit3), and YER178W (Δpda1)) were purchased from Open Biosystems (Huntsville, AL). Yeast extract and tryptone were purchased from Teknova (Hollister,

CA). Glucose and HEPES were purchased from Amresco (Solon, OH). 2-13C propionate and 2-13C pyruvate were purchased from Cambridge Isotope Laboratories (Andover,

MA). Coenzyme A (CoA), D2O, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), propionic anhydride and oxaloacetate were purchased from Sigma (St. Louis, MO). Acetic anhydride was a gift from Dr. Richard T. Taylor (Miami University, Oxford, OH). DNA primers for PCR and DNA sequencing were purchased from Integrated DNA Technologies (Coralville, IA). The pTrcHis TOPO vector was purchased from Invitrogen (Carlsbad, California), while the pGroESL plasmid was kindly supplied by Dr. Anthony Gatenby (Central Research and Development, DuPont). Tryptic soy broth (TSB) was purchased from Becton, Dickinson and Company (Sparks, MD). Isopropyl-β-D- thiogalactopyranoside (IPTG) was purchased from Gold Bio Technology (St. Louis, MO). Ni-NTA Agarose was purchased from QIAGEN (Valencia, CA), while Ni-CAM HC Resin was purchased from Sigma (St. Louis, MO). Bio-Rad protein assay reagent was purchased from Bio-Rad Laboratories (Hercules, CA). All other reagents were purchased from either Sigma (St. Louis, MO) or Fisher Scientific (Fair Lawn, New Jersey). 2.2.2 Methods 2.2.2.1 Comparison of amino acid sequences. The amino acid sequence of Cit3p, a citrate synthase homologue in S. cerevisiae, (gi|1314076) was used for pair- wise alignments to other citrate synthase homologues. These included two other citrate synthase homologues in S. cerevisiae (Cit2p (gi|1907148) and Cit1p (gi|1302469)), two higher eukaryotic citrate synthases (Sus scrofa, (gi|47523618) and Gallus gallus, (gi|1311373)), and the citrate synthase and a methylcitrate synthase of E. coli (GltA (gi|3025888) and PrpC (gi|1648967) respectively). Alignments were performed using BLASTP with a BLOSUM62 matrix and the default settings for BLASTP (at http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). If at least three sequences had the same amino acid at any given position, this amino acid was shaded black. If at least three sequences had structurally similar amino acids at any given position, these amino acids were shaded gray. Similarity was based on the following amino acid groups: STC, GA, KR, ED, FYW, and LVI. These pair-wise alignments were combined in a single comparison to determine percent identity and percent similarity. Percent identity was calculated to be the number of identical amino acids divided by the total number of

amino acids in the sequence that was being compared. Percent similarity was calculated to be the number of identical amino acids plus the number of similar amino acids, defined by the rules already mentioned, divided by the total number of amino acids in the sequence being compared. 2.2.2.2 Cloning of CIT1 and CIT3 cDNAs. The CIT1 and CIT3 genes were PCR amplified from yeast genomic DNA (Table 2-1). The resulting PCR products were analyzed by agarose gel electrophoresis, then ligated into the pTrcHis TOPO vector, and plated on TSB agar with 50 μg/mL ampicillin. Colonies were screened using Express Forward primer and a reverse primer (Table 2-1) of either CIT1 or CIT3 to verify positive directional cloning with the correct orientation for expression. A plasmid encoding a molecular chaperone, pGroESL, was co-transfected with CIT3. Cells

containing the CIT3 construct were made competent using standard methods with CaCl2. To these cells 1 μg of pGroESL was added followed by incubation on ice for 30 minutes. The cells were then heat shocked at 42 oC for 45 seconds. A 500 μL protion of TSB was added and co-transfected cells were recovered by shaking at 37 oC for 40 minutes followed by incubation on TSB agar with 50 μg/mL ampicillin and 50 μg/mL chloramphenicol. 2.2.2.3 Cit1p over-expression and purification. A 5 mL culture containing 50 μg/mL ampicillin was grown overnight in a shaking incubator at 37 oC in TSB media. The 5 mL culture was added to 1L of fresh TSB with 50 μg/mL ampicillin, o grown at 37 C to an OD600 of 0.4 - 0.6 when IPTG (0.75 mM final concentration) was added and incubation continued overnight at 37 oC. The 1 L culture was centrifuged at 4200 x g for 30 minutes at 4 oC and the cell pellet was resuspended in 15 mL of column

buffer (5 mM imidazole, 0.5 M NaCl, 50 mM NaPO4) plus 10 mM β-mercaptoethanol and 0.5% Tween 20. The sample was sonicated using a Fisher Scientific Sonic Dismembrator Model 100, centrifuged (12000 x g, 4 oC, 30 minutes), and the supernatant was immediately loaded onto 2 mL of Ni-NTA Agarose packed column. Unbound proteins were removed with 3 column volumes of column buffer with 20 mM imidazole plus 10 mM β-mercaptoethanol, followed by washing with column buffer containing 40 mM imidazole until no protein was detected using Bio-Rad protein assay reagent. Cit1p was eluted with column buffer containing 250 mM imidazole and 1 mL elution fractions

were collected. Protein concentration was determined spectrophotometrically at 595 nm using the Bio-Rad protein assay reagent. Cit1p was analyzed for purity using a Laemmli 12.5% SDS-PAGE gel and visualized with Coomassie blue (18). The identity of Cit1p was confirmed by excising from the gel, digesting with trypsin, running on a MALDI- TOF MS, and matching expected tryptic digest fragments (19). 2.2.2.4 Cit3p over-expression and purification. Cit3p was purified from three liters of culture (co-transfected with pGroESL) using separate one liter cultures on three consecutive days. For each of these preparations, a 5 mL culture with 50 μg/mL ampicillin and 50 μg/mL chloramphenicol was grown overnight at 37 oC in TSB. The 5 mL culture was added to 1L of freshly-prepared TSB containing 50 μg/mL ampicillin and o 50 μg/mL chloramphenicol, grown at 37 C to an OD600 of 0.4 – 0.6 when IPTG (0.75 mM final concentration) was added, and incubation was continued overnight at 37 oC. The 1 L culture was centrifuged at 4200 x g for 30 minutes at 4 oC, and the pellet was

resuspended in 15 mL of column buffer (10 mM imidazole, 0.3 M NaCl, 50 mM NaPO4) plus 10 mM β-mercaptoethanol and 0.5% Tween 20. The sample was sonicated using a Fisher Scientific Sonic Dismembrator Model 100, centrifuged at 12000 x g for 30 minutes at 4 oC, and the supernatant was immediately loaded onto 2 mL of Sigma Ni- CAM HC resin packed column. Unbound protein was removed with 3 column volumes of column buffer containing 20 mM imidazole plus 10 mM β-mercaptoethanol, followed by column buffer containing 40 mM imidazole until no protein was detected using Bio- Rad protein assay reagent. Cit3p was then eluted from the column using column buffer containing 250 mM imidazole and collected in 1 mL fractions. Protein concentration was determined spectrophotometrically at 595 nm using the Bio-Rad protein assay reagent. Cit3p was analyzed for purity using a Laemmli 12.5% SDS-PAGE gel and visualized with Coomassie blue (18). The identity of Cit3p was confirmed by excising from the gel, digesting with trypsin, running on a MALDI-TOF MS, and matching expected tryptic digest fragments (19). 2.2.2.5 Coenzyme A Thio-ester synthesis. Propionyl-CoA and acetyl- CoA were synthesized using propionic anhydride or acetic anhydride, respectively. A three-fold molar ratio of the appropriate anhydride was added drop wise to a solution of CoA in 0.2 M phosphate buffer pH 8.0 and stirred on ice for 30 minutes. To test for

completion of the reaction 5 μL of the CoA / anhydride solution was added to 0.15 mM DTNB. A yellow color indicated the presence of free CoA. If CoA was not completely reacted, additional anhydride was added and the mixture was stirred on ice for another 30 minutes. This process was repeated until no yellow color was observed. The solution was adjusted to pH < 3 with HCl and extracted 3 times with water-saturated ether to remove the organic acids. The water layer, containing the acyl-CoA product, was then rotary evaporated to dryness. The sample was resuspended in 1 mL of 50 mM HEPES buffer (pH 7.5), 0.1 M KCl, and 0.54 M glycerol. Concentration of the CoA product was determined spectrophotometrically at 260 nm using a molar extinction coefficient of 16,800 M-1 cm-1 (20). Integrity of the acyl-CoA products were analyzed by LC-MS as previously described (20). 2.2.2.6 Enzyme assay conditions for measurement of citrate and methylcitrate synthase activities. Spectrophotometric quantification of acyl-CoA consumption was determined by measuring the free CoA produced in the reaction mixture using DTNB. The TNB- anion forms when CoA reacts with DTNB. This reaction can be monitored since the anion absorbs at 412 nm with an extinction coefficient of 14,150 M-1 cm-1. One mL reaction mixtures contained 50 mM HEPES buffer (pH 7.5), 0.1 M KCl, 0.54 M glycerol, 0.05 mM oxaloacetate, and saturating amounts of CoA substrate were used in these studies. Saturation with the acyl-CoA substrates was confirmed by analysis of replicate reactions with increasing concentrations of the corresponding CoA thioester. For these assays, the buffer and substrate were incubated in 1.5 mL cuvettes at 37 oC using a Cary 1E spectrophotometer (Varian) equipped with a circulating water jacket. Prior to addition of the enzyme, reaction mixtures were monitored for 3 – 5 minutes to establish a base line of absorbance at 412 nm. Once a baseline was established recombinant Cit1p, Cit3p, or cell extract was added, and the reaction was monitored for an additional 10 minutes at 412 nm. Data were analyzed using the IGOR software program (WaveMetrics, Inc.). 2.2.2.7 Wild-type, Δcit1, Δcit3 sample preparation for native enzyme activity analysis. A 5 mL culture of either WT, Δcit1, or Δcit3 was grown overnight in a shaking incubator at 30 oC in either 1% yeast extract, 2% tryptone, 1% dextrose (YPD), or 1% yeast extract, 2% tryptone, 2% glycerol (YPG), or 1% yeast extract, 2% tryptone,

2% ethanol (YPE) medium. The 5 mL culture was added to 200 mL of fresh medium o (either YPD, YPG, or YPE) and grown at 30 C until OD600 reached 1.0. A 40 mL portion of these cultures was centrifuged (12000 x g at 4 oC for 30 minutes) and the resulting pellets were stored at -80 oC prior to analysis. Immediately before enzyme analysis, samples were thawed and resuspended in 3 mL of 50 mM HEPES buffer (pH 7.5) plus 0.5% Tween 20. Glass beads were added and the sample was vortexed at 1 minute bursts 6 times. The glass beads were filtered, supernatant collected, and centrifuged as before to remove cell debris. These samples were used immediately for enzyme assays. Protein concentration was determined spectrophotometrically at 595 nm using the Bio-Rad protein assay reagent. 2.2.2.8 Preparation of yeast samples for 13C-NMR analysis. Sample preparation followed the methods of Escalante-Semerena and Horswill (11). A 5 mL culture of WT, Δcit1, or Δcit3 was grown overnight in a shaking 30 oC incubator in 1% YPD medium, added to 500 mL of fresh YPD, and then grown for 24 hours at 30 oC. After 24 hours the cells were centrifuged at 4 oC for 30 minutes at 4200 x g, supernatant removed, and the cell mass was resuspended in 50 mL YPD containing one of the following: 5 mM 2-13C labeled propionate or 5 mM 2-13C labeled pyruvate. These culture were incubated for 45 minutes in a shaking 30 oC incubator and then centrifuged as above. The culture supernatant was removed and the cell mass resuspended in 50 mL of 10X phosphate-buffered saline (PBS) (pH 7.4, 1.37 M NaCl, 27 mM KCl, 43 mM

Na2HPO4, 14 mM KH2PO4). Perchloric acid (final concentration 4%) was added to the suspension and the sample was stored at -80 oC until analysis by 13C-NMR. 2.2.2.9 Analysis by 13C-NMR. Frozen samples were thawed at room temperature and cell debris removed by centrifugation at 4 oC for 30 minutes at 4200 x g. The pH of the supernatant was then adjusted to 7.0 with 10 M KOH, the mixture was centrifuged for 30 minutes at 4 oC at 12000 x g to remove the salt precipitate, and the resulting supernatant was then lyophilized. The lyophilized powder was resuspend in 5 13 mL of 10% D2O. C-NMR spectra were acquired at 75.5 MHz with a deuterium lock on a Bruker Instrument DPX-300 Avance console with a 7.05-T narrow-bore magnet. 13C- NMR proton-decoupled spectra were obtained with a 30o pulse angle, relaxation time of 2s, 10,000 scans, under no temperature control, and the spectra were Fourier-transformed

with 1-Hz line broadening. Chemical shifts presented in this work were calibrated to that 13 of CDCl3 set at 77.7 ppm and 2- C propionate set at 30.6 ppm. 2.2.2.10 GC-MS examination of isobutanol in wild-type and Δcit3. Sample preparation was conducted as described by Eden et al. with some modification (21). Briefly, a 5 mL culture of WT or Δcit3 was grown overnight in a shaking 30 oC incubator in YPD medium. Then 1 mL was added to 50 mL of YPD medium with 5 mM pyruvate, it was grown for 24 hours at 30 oC. After 24 hours a 1 mL sample was collected. The 1 mL sample was centrifuged at 4 oC for 30 minutes at 20,000 x g to remove yeast cells and the supernatant was collected. Then, 10 μL of the supernatant was injected into a Varian CP-3800 GC-MS with a 30-meter (0.25 mM inner diameter) WCOT fused silica capillary column and conditions were followed as described by Dickinson et al. with some modification (13,15,26). Briefly, the injector temperature was set at 250 oC followed by a 60 oC isothermal run for ten minutes, split ratio of 5, and a helium carrier gas flow rate of 1 mL/min with a column pressure of 8 psi. Iso-amyl alcohol was used as an internal standard and a ratio of peak areas was used to quantify the amount of isobutanol. 2.3 Results and Discussion 2.3.1 Structural sequence comparison. Cit3p in a previous study was identified as a citrate synthase homologue by comparing sequence homology of a then unknown ORF to Cit2p and Cit1p (6). Our work and a previous study by Jia et al. showed that Cit3p and Cit1p were about 50% homologous (6). In addition we compared Cit3p to other known citrate synthases from higher eukaryotes (pig and chicken) (Figure 2-1). Sequences from pig and chicken were chosen because these enzymes have been thoroughly characterized and because X-ray crystal structures have been reported. CIT1 displayed the highest percent similarity to each of the eukaryotic sequences at about 60%, while Cit3p showed about 50% similarity (Table 2-2). Both Cit1p and Cit3p had higher percent similarity to the pig enzyme than the chicken enzyme. We wanted to know if a sequence comparison between Cit1p and Cit3p to a known methylcitrate synthase in E. coli, PrpC, could provide clues about the possible identity of either Cit1p or Cit3p as a methylcitrate synthase (Figure 2-1). Our analysis indicated that Cit1p has about 29% similarity, while Cit3p had about 26% similarity to both citrate synthase (GltA) and

methylcitrate synthase (PrpC) of E. coli. Neither Cit1p nor Cit3p showed dramatic differences in identity or similarity to either GltA or PrpC indicating that the amino acid sequence homology alone could not provide strong evidence for either Cit1p or Cit3p being a specific methylcitrate synthase in S. cerevisiae. This comparison does, however, show that the yeast Cit proteins are more closely related to those of higher eukaryotes even though propionate metabolism in yeasts occurs by the same mechanism as it does in bacteria. 2.3.2 Cloning of CIT1 and CIT3 cDNAs. The PCR amplification, cloning and verification of proper orientation into the pTrcHis TOPO vector was straight-forward and achieved with no obstacles encountered. Agarose gel electrophoresis demonstrated cDNAs of the expected size for both CIT1 and CIT3. Cloning of these cDNAs into the pTrcHis TOPO vector was shown to be successful by diagnostic PCR analysis with approximately 50% of colonies having plasmids with the correct size and orientation of the cDNA inserts. 2.3.3 Expression and purification of Cit1p and Cit3p. Initial attempts to over- express and purify Cit1p and Cit3p using Sigma Ni-CAM HC Resin followed a protocol similar to that described in the Methods but with less imidazole and no β- mercaptoethanol as suggested by Sigma. Purified in this way, Cit1p showed activity with acetyl-CoA and no activity with propionyl-CoA, while Cit3p showed no activity with either substrate (data not shown). Examination of the amino acid sequence showed 7 cysteine residues in Cit3p and 1 cysteine residue in Cit1p. A second attempt was made to over-express and purify both Cit1p and Cit3p including β-mercaptoethanol as an antioxidant in all steps except the last washing before the elution with 250 mM imidazole. β-Mercaptoethanol is known to react with DTNB and washing without β- mercaptoethanol in the next to last step eliminated background interference with the enzyme assay that uses DTNB to measure reduced CoA thiol. Purified in this way, Cit1p showed activity with acetyl-CoA and no activity with propionyl-CoA while Cit3p showed activity with both acetyl-CoA and propionyl-CoA (data not shown). The purity of these preparations was then analyzed on 12.5% SDS PAGE gels and this analysis indicated the presence of a number of contaminating proteins. A third attempt was made to over-express and purify both Cit1p and Cit3p using a Ni-NTA Agarose from Qiagen.

Purity was ascertained for Cit1p on a 12.5% SDS PAGE gel and was noted to be about 90% pure (Figure 2-2A) and activity for each acyl-CoA was the same as previously mentioned (data not shown). Cit3p expression resulted in a poor yield and was improved by adding the plasmid pGroESL. We then optimized the Sigma Ni-CAM HC Resin suggested protocol, as described in Methods, and increased the amount of starting culture to three liters. The yield for Cit3p was consistently lower than that for Cit1p and we postulated that the over-expression of Cit3p using the pTrcHis TOPO vector was toxic to the cells. This observation was based on repeated uses of a continually grown seed starter culture that demonstrated decreasing yield upon repeated induction and purification of Cit3p. Yields were higher when a fresh starter culture was used every time. When using a fresh seed culture for each purification and three liters of culture the yield upon combination of all fractions was enough to perform our experiments. The resulting Cit3p preparation was determined to be about 90% pure when examined on a 12.5% SDS PAGE gel (Figure 2-2B). 2.3.4 Substrate specificities of Cit1p and Cit3p. Analysis of the citrate synthase and methylcitrate synthase activities of the purified Cit1p showed that this enzyme is a highly specific citrate synthase, catalyzing the condensation of oxaloacetate with acetyl- CoA but not with propionyl-CoA. No activity with propionyl-CoA was observed even at high concentrations. In contrast, Cit3p appeared to be an enzyme with dual specificity catalyzing the condensation of oxaloacetate with either acetyl-CoA or propionyl-CoA. Kinetic analyses were performed to compare the substrate specificities of these enzymes

with their acyl-CoA substrates (Table 2-3). The apparent Km of Cit1p with acetyl-CoA

was determined to be 76 μM. The apparent Km of Cit3p with acetyl-CoA was determined to be 1200 μM while the apparent Km with propionyl-CoA was 520 μM. Data obtained by Escalante-Semerena and Horswill during their study of the

methylcitrate synthase of S. typhimurium determined Km values of 285 μM and 48 μM with acetyl-CoA and propionyl-CoA, respectively (11). Thus, Cit3p appears to have methylcitrate synthase activity but differs from the more specific bacterial methylcitrate

synthase. In our comparison of Cit1p and Cit3p, our calculated kcat values showed that both had the same turnover number with acetyl-CoA at 11 s-1. Propionyl-CoA with -1 Cit3p exhibited a 2-fold lower kcat of 5.4 s . The catalytic efficiency (kcat / Km) of Cit3p

with both acetyl-CoA and propionyl-CoA was very similar at 9 and 10x103 M-1 s-1, respectively. The catalytic efficiency of Cit1p with acetyl-CoA was slightly more than an order of magnitude greater at 150x103 M-1 s-1. These data support a metabolic function of Cit3p in metabolism of propionyl-CoA and for Cit1p as a specific citrate synthase for metabolism of acetyl-CoA. 2.3.5 Effect of gene knockouts on native citrate and methylcitrate synthase activities. The availability of mutant strains lacking specific genes in S. cerevisiae allows functional studies through examination of the effects of such “knock-out” mutations. Therefore we decided to examine the citrate synthase and methylcitrate synthase activities resulting from native enzymes in Δcit1, Δcit3, and wild-type strains. S. cerevisiae under certain media conditions is known to either repress or activate the expression of different genes. We studied the native citrate synthase and methylcitrate synthase activities of each strain using three different growth media: YPD, YPG, and YPE. Previous studies of CIT1, CIT2, and CIT3 transcript levels showed that when CIT1 was knocked out CIT3 mRNA was increased (6). Our data (Table 2-4) support this observation at the level of enzyme activity because activity with acetyl-CoA and with propionyl-CoA increased significantly in cell-free extracts of a Δcit1 strain. This increase was independent of the growth medium used. Although both the citrate and methylcitrate synthase activities increased dramatically in the Δcit1 strain, the ratio of these two activities decreased, indicating a decrease in activity of citrate synthase compared to methylcitrate synthase. This observation supports the identity of Cit3p as a methylcitrate synthase. However, deletion of CIT3 had little effect on the levels of either citrate or methylcitrate synthase activity, nor on the ratio of these two activities in cell free extracts. This observation suggests that another enzyme, perhaps Cit2p, may also have methylcitrate synthase activity. Cit2p is known to be peroxisomal and so could participate in the catalysis measured in our cell free extracts along with the mitochondrial enzymes. In general, these data help to support the identification of Cit3p as a mitochondrial methylcitrate synthase. They also indicate that another methylcitrate synthase likely exists and that the regulation of these enzymes’ expression may be complex.

2.3.6 Propionate metabolism in wild-type and mutant strains. 2-13C labeled propionate was used in an attempt to address the first important reaction that a methylcitrate synthase would catalyze, the condensation between propionyl-CoA and oxaloacetate to make 2-methylcitrate in the 2-methylcitric acid cycle. Figure 2-3 shows spectra of wild-type S. cerevisiae incubated either with or without exogenously added sodium 2-13C propionate. Under our rich medium conditions, some signal from unlabeled carbon (natural abundance) is observed, and the only difference between the two spectra is a peak around 30.6 ppm that is the 2-13C labeled propionate peak. Data (not shown) were collected for the Δcit1 and Δcit3 strains, and their spectra look similar to that of the wild-type in that the only peak difference between added and not added 2- 13C labeled propionate is the peak around 30.6 ppm. Prior work by Escalante-Semerena and Horswill showed NMR spectra of S. typhimurium and assigned peaks in their NMR spectra to metabolites such as 2-methylcitate around 49 ppm and 2-methyl-cis-aconitate around 141 ppm along with propionate around 30.6 ppm (11). We suspect that because both Δcit1 and Δcit3 strains have activity with acetyl-CoA that we see no accumulation of methylcitrate intermediates because metabolism of propionate via the 2-methylcitrate pathway would result in pyruvate entering into the TCA cycle and the labeled carbon 13 accumulating as CO2. No specific conclusions can be drawn from these data regarding the identity of CIT1 or CIT3 as the methylcitrate synthase. 2.3.7 Pyruvate metabolism in wild-type and mutant strains. We next characterized the two citrate synthases, Cit1p and Cit3p, by NMR using the knockout strains and an entry point into the TCA cycle, 2-13C labeled pyruvate. Figure 2-4 shows NMR spectra resulting from the addition of 2-13C labeled pyruvate to either wild-type or Δcit3 which were compared to spectra where 2-13C labeled pyruvate was not added (Figure 2-3B for wild-type and data not shown for Δcit3). As with 2-13C-propionate, we concluded that no difference exists in a wild-type strain with the addition of 2-13C labeled 13 pyruvate most likely because pyruvate is completely metabolized to CO2. Yet for Δcit3 (Figure 2-4B) we observed the accumulation of one spectral peak with a chemical shift at 68 ppm, which is consistent with the known chemical shift of isobutanol (C1). A dramatic increase in labeling of acetate (C1) at 181 ppm was also observed. There was also in an increase in carboxylate peaks and methylene peaks (10-25 ppm) that are not

conclusively assigned. The unidentified carboxylate peaks may correspond to glutamate carboxylates (182 and 174 ppm). Accumulation of each of these compounds could occur if pyruvate entry into the TCA cycle, catalyzed by pyruvate dehydrogenase, was inhibited and diverted to different metabolic routes (Figure 2-5). Prior work in either the fungi Aspergillus fumigatus or in E. coli has shown that accumulation of propionyl-CoA inhibits pyruvate dehydrogenase (PDH) and CoA-dependent enzymes (22,23). Thus, we propose that these observed alterations in pyruvate metabolism in the Δcit3 strain resulted from inhibition of PDH by propionyl-CoA which may accumulate in this strain if Cit3p is a methylcitrate synthase. Aside from entry into the TCA cycle, pyruvate can be converted to branched-chain acids and alcohols through the action of acetolactate synthase, a thiamine pyrophosphate dependent enzyme. This would account for the observation of labeled isobutanol. Work by Dickinson et al. using NMR Spectroscopy to examine metabolism of valine and leucine in S. cerevisiae showed that isobutanol accumulated from valine and had a chemical shift around 68 ppm (C1) (13,14). There is also a well established “PDH by-pass” utilizing pyruvate decarboxylase and aldehyde dehydrogenase to produce acetate (24). This would explain our observed increase in acetate in the presence of PDH inhibition. These data indirectly suggest that CIT3 is a methylcitrate synthase that metabolizes propionyl-CoA via the 2-methylcitric acid pathway because propionyl-CoA is known to inhibit the E2 subunit of the pyruvate dehydrogenase complex. Inhibition of the pyruvate dehydrogenase complex would result in pyruvate entry into the TCA cycle being diverted and our data shows an accumulation of isobutanol and acetate only in a Δcit3 strain and not in wild-type (Figure 2-4) or Δcit1 strains. We hypothesized from the data obtained with Δcit3 that propionyl-CoA accumulating in this strain inhibits PDH (through the E2 subunit). We further investigated effects of PDH complex by examining pyruvate metabolism in a Δpda1 strain lacking the E1 alpha subunit of the PDH complex. The absence of a E1 alpha subunit would be expected to structurally destabilize the PDH complex (25). Although this would be different from inhibition of E2, it would represent the effect of complete loss of PDH activity, whereas inhibition of E2 could occur in the presence of dehydrogenase activity catalyzed by E1. Our data showed that unlike the Δcit3 strain the

Δpda1 strain did not accumulate isobutanol (Figure 2-6). There appeared to have been an increase in acetate but, this was not as dramatic as in the Δcit3 strain. There was also in an increase in carboxylate peaks that are not conclusively assigned but may correspond to glutamate carboxylates (182 and 174 ppm). This would indicate pyruvate entry into the TCA cycle through the PDH bypass rather than through PDH (24). These results are consistent with our hypothesis regarding the differences in inhibition of PDH E2 versus complete loss of PDH complex. 2.3.8 Analysis of isobutanol by GC-MS. We used GC-MS to confirm the presence of isobutanol by examining both the wild-type and Δcit3 strains. Isobutanol has a mass of 74 and the most abundant fragment produced is an M+1 from the loss of water having an m/z value of 57. We examined the specific ion chromatograph corresponding to this fragment and the associated mass spectra, compared to standard isobutanol. Our results indicated the presence of isobutanol in the Δcit3 strain observed as a distinct peak with an elution profile comparable to that of standard isobutanol (Figure 2-7). Integration of the peak corresponding to m/z 57 compared to standard isobutanol determined a concentration of 65 +/- 3 μg/mL. A peak corresponding to isobutanol was not observed in the wild-type strain and any isobutanol produced during growth of the wild-type strain in YPD medium was below our limit of detection. Previous studies examining isobutanol production utilized minimal media with branched-chain amino acids as the sole nitrogen source (13,15). The data obtained supports that in the Δcit3 strain pyruvate metabolism was diverted to a route of isobutanol production in a rich medium containing many carbon and nitrogen sources. This data is consistent with our observation using NMR spectroscopy. 2.4 Conclusions We conclude that CIT3 is the gene coding for the methylcitrate synthase of S. cerevisiae based on three observations. Analysis of the citrate synthase and methylcitrate synthase activities of the purified Cit1p and Cit3p showed that Cit1p is a highly specific citrate synthase while Cit3p is a dual specific citrate and methylcitrate synthase. The second observation was a decrease in the ratio of native citrate and methylcitrate synthase activities in Δcit1. This observation is in agreement with the activities measured in the recombinant enzymes. The third observation was an increase of isobutanol and acetate

observed by NMR spectroscopy after the addition of 2-13C pyruvate to the Δcit3 strain. It is hypothesized that the Δcit3 strain accumulates propionyl-CoA which inhibits the E2 subunit of the pyruvate dehydrogenase complex. The identification of the methylcitrate synthase allows for further study into why bacteria but not yeasts are inhibited by propionic acid and is of value to other laboratories using metabolic methods with S. cerevisiae.

Cit3p 1 MVQRLLPGAHICRRSFNSSAIIKSSALTLKEALENVIPKK Cit2p 1 MTVPYLNSNRNVASYLQSNSSQEKTLKERFSEIYPIH Cit1p 1 MSAILSTTSKSFLSRGSTRQCQNMQKALFALLNARHYSSASEQTLKERFAEIIPAK PIGa 1 MALLTAAARLFGAKNASCLVLAARHASASSTNLKDILADLIPKE CHICKb 1 ASSTNLKDVLASLIPKE GltAc 1 MADTKAKITL

Cit3p 41 RDAVKKLKACYGSTFVGPITISSVLGGMRGNQSMFWQGTSLDPEHGIKFQGLTIEE Cit2p 38 AQDVRQFVKEHGKTKISDVLLEQVYGGMRGIPGSVWEGSVLDPEDGIRFRGRTIAD Cit1p 57 AEEIKKFKKEHGKTVIGEVLLEQAYGGMRGIKGLVWEGSVLDPEEGIRFRGRTIPE PIG 45 QARIKTFRQQHGNTVVGQITVDMMYGGMRGMKGLVYETSVLDPDEGIRFRGYSIPE CHICK 18 QARIKTFRQQHGNTAVGQITVDMSYGGMRGMKGLIYETSVLDPDEGIRFRGFSIPE GltA 11 TGDTTIELDVLKGTLGQDVIDIRSLGSKGVFTFDPGFTSTASCESKITFIDGDEGI PrpCd 1 MTDTTILQNNTHVIKPKKSVALSGVPAGNTALCTVGKSGND

Cit3p 97 CQNRLPNTGIDGDNFLPESMLWLLMTGGVPTFQQAASFRKELAIRGRKLPHYTEKV Cit2p 94 IQKDLPKAK-GSSQPLPEALFWLLLTGEVPTQAQVENLSADLMSRS-ELPSHVVQL Cit1p 113 IQRELPKAEGSTE-PLPEALFWLLLTGEIPTDAQVKALSADLAARS-EIPEHVIQL PIG 101 CQKMLPKAK-GGEEPLPEGLFWLLVTGQIPTEEQVSWLSKEWAKRAA-LPSHVVTM CHICK 74 CQKLLPKAG-GGEEPLPEGLFWLLVTGQIPTPEQVSWVSKEWAKRAA-LPSHVVTM GltA 67 LLHRGFPIDQLATDSNYLEVCYILLYGEKPTQEEYDEFRTTVT-RHTMIHEQITRL PrpC 42 LHYRGYDILDLAEHCEFEEVAHLLIHGKLPTRDELNAYKSKLKALRG-LPANVRTV

Cit3p 153 LSSLPKDMHPMTQLAI---GLASMNKGSLFATNYQKGLIGKMEFWKDTLEDSLNLI Cit2p 148 LDNLPKDLHPMAQFSI---AVTALESESKFAKAYAQG-ISKQDYWSYTFEDSLDLL Cit1p 167 LDSLPKDLHPMAQFSI---AVTALESESKFAKAYAQG-VSKKEYWSYTFEDSLDLL PIG 155 LDNFPTNLHPMSQLSA---AITALNSESNFARAYAEG-IHRTKYWELIYEDCMDLI CHICK 128 LDNFPTNLHPMS QLSA---AITALNSESNFARAYAEG-INRTKYWEFVYEDAMDLI GltA 122 FHAFRRDSHPMAVMCGITGALAAFYHDSLDVNNPRHREIA------AFRLL PrpC 97 LEALPAASHPMDVMR---TGVSAL--GCTLPEKEGHTVSGARDI-ADKLLASLNSI

Cit3p 206 ASLPLLTGRIYSNITNEGHPLGQYSEEVDWCTNICSLLGMTNGTNSSNTCNLTSQQ Cit2p 200 GKLPVIAAKIYRNVFKDGK-MGEVDPNADYAKNLVNLIG------SK Cit1p 219 GKLPVIASKIYRNVFKDGK-IDTSTPN ADYGKN LAQLLGY ENK------PIG 207 AKLPCVAAKIYRNLYREGSSIGAIDSKLDWSHNFTNMLGYTDA------CHICK 180 AKLPCVAAKIYRNLYRAGSSIGAIDSKLDWSHNFTNMLGYTDP------GltA 167 SKMPTMAAMCYKY--SIGQPFVYPRNDLSYAGNFLNMMFST--PCETYEVNPVLER PrpC 147 ----LLYWYHYSH---NGERIQPETDDDSIGGHFLHLLHGEKPTQS------

Cit3p 262 SLDFIN LMRLYTG IHV DHEGGNVSAHTTHLVGSALSDPYLSYSSGIM GLAGPLHGL Cit2p 240 DEDFVDLMRLYLTIHSDHEGGNVSAHTSHLVGSALSSPYLSLASGLNGLAGPLHGR Cit1p 261 --DFIDLMRLYLTIHSDHEGGNVSAHTTHLVGSALSSPYLSLAAGLNGLAGPLHGR PIG 250 --QFTELMRLYLTIHSDHEGGNVSAHTSHLVGSALSDPYLSFAAAMNGLAGPLHGL CHICK 223 --QFTELMRLYLTIHSDHEGGNVSAHTSHLVGSALSDPYLSFAAAMNGLAGPLHGL GltA 219 AMDRILIL------HADHEQ-NASTSTVRTAGSSGANPFACIAAGIASLWGPAHGG PrpC 186 ---WEKAMHISLVLYAEHEF-NASTFTSRVIAGTGSDVYSAIIGAIGALRGPKHGG

Cit3p 318 AAQEVVRFLIEMNS--NISSIAREQEIKDYLWKILNSNRVIPGYGHAVLRKPDPRF Cit2p 296 ANQEVLEWLFALKE--EVNDDYSKDTIEKYLWDTLNSGRVIPGYGHAVLRKTDPRY Cit1p 315 ANQEVLEWLFKLRE--EVKGDYSKETIEKYLWDTLNAGRVVPGYGHAVLRKTDPRY PIG 304 ANQEVLVWLTQLQK--EVGKDVSDEKLRDYIWNTLNSGRVVPGYGHAVLRKTDPRY CHICK 277 ANQEVLLWLSQLQK--DLGADASDEKLRDYIWNTLNSGRVVPGYGHAVLRKTDPRY GltA 268 ANEAALKMLEEISSVKHIPEFVRRAKDKNDSFRLM------GFGHRVYKNYDPRA PrpC 238 ANEV---SLEIQQ---RYET---PDEAEADIRKRVENKEVVIGFGHPVYTIADPRH

Cit3p 372 TAMLEFAQKRPIEFENDKNVLLMQKLAEIAPKVLLEHGKSKNP------FPNVDS Cit2p 350 MAQRKFAMDHFPDYELFK---LVSSIYEVAPGVLTEHGKTKNP------WPNVDA Cit1p 369 TAQREFALKHFPDYELFK---LVSTIYEVAPGVLTKHGKTKNP------WPNVDS PIG 358 TCQREFALK---HLPHDPMFKLVAQLYKIVPNVLLEQGKAKNP------WPNVDA CHICK 331 TCQREFALK---HLPSDPMFKLVAQLYKIVPNVLLEQGKAKNP------WPNVDA GltA 317 TVMRETCH------EVLKELGTKDDLLEVAME--LEHIALNDPYFIEKKLYPNVDF PrpC 285 QVIKRVAK----QLSEEGGSLKMYHIADRLETVMWE---TKKM------FPNLDW

Cit3p 421 ASGILFYHYGIRELLFFTVIFGCSRAMGPLTQLVWDRILGLPIERPKSLNLEGLEA Cit2p 396 HSGVLLQYYGLKESSFYTVLFGVSRAFGILAQLITDRAIGASIERPKSYSTEKYKE Cit1p 415 HSGVLLQYYGLTEASFYTVLFGVARAIGVLPQLIIDRAVGAPIERPKSFSTEKYKE PIG 404 HSGVLLQYYGMTEMNYYTVLFGVSRALGVLAQLIWSRALGFPLERPKSMSTDGLIK CHICK 377 HSGVLLQYYGMTEMNYYTVLFGVSRALGVLAQLIWSRALGFPLERPKSMSTAGLEK GltA 365 YSGIILKAMGIPSSM-FTVIFAMARTVGWIAHWNEMHTDGMKIARPRQLYTGYDKR PrpC 327 FSAVSYNMMGVPTEM-FTPLFVIARVTGWAAHIIEQRQDNKII-RPSANYTGPEDR

Cit3p 477 LKT ASNVNKL Cit2p 452 LVKNIESKL Cit1p 470 LVKKIESKN PIG 460 LVDSK CHICK 433 LSAGG GltA 420 DFKSALKR PrpC 381 PFVSIDDRC

Figure 2-1: Amino acid sequence comparison of citrate synthase and a methylcitrate synthase homologues. Sequences were obtained from NCBI and aligned using BLASTP as described above. a, Sus scrofa. b, Gallus gallus. c, citrate synthase in E. coli. d, methylcitrate synthase in E. coli.

A. 1 2 B. 1 2 212 212 116 116 97.4 97.4 66.2 66.2 57

57 40

38 38

14.4

Figure 2-2: SDS PAGE gel electrophoretic analysis of recombinant Cit1p and Cit3p. (A) 1, molecular weight markers. 2, Cit1p (calculated mass 57 kDa). (B) 1, molecular weight markers. 2, Cit3p (calculated mass 57 kDa). Molecular weight markers (Amresco Wide Range Protein Marker): 212, 116, 97.4, 66.2, 40, 38, 21, 14.4 kDa.

A. Propionate

Figure 2-3: 13C-NMR spectra of wild-type S. cerevisiae in YPD with 2-13C- propionate. (A) Spectrum of a sample with 2-13C propionate added. (B) Spectrum of a sample where propionate was not added (natural abundance).

Acetate

B. Isobutanol

Acetate

Figure 2-4: 13C-NMR spectra of S. cerevisiae grown in YPD with 2-13C pyruvate. (A) Spectrum of wild-type S. cerevisiae. (B) Spectrum of Δcit3 S. cerevisiae.

Cytosol Mitochondria Propionyl-CoA Cit3p

2-Methylcitrate

Δpda1 Δcit3 Δcit3 Acetolactate Pyruvate Pyruvate Δpda1 Δcit3 Δcit3 Valine Acetyl-CoA Acetaldehyde Cit1p or Cit3p Δpda1 Δcit3 Δcit3

Isobutanol TCA Acetate Cycle

Figure 2-5: Model of pyruvate flux in wild-type and mutant Saccharomyces cerevisiae.

Acetate

Figure 2-6: 13C-NMR spectra of Δpda1 S. cerevisiae grown in YPD with 2-13C pyruvate. (C) Spectrum of a sample with 2-13C pyruvate added. (B) Spectrum of a sample where pyruvate was not added (natural abundance).

Figure 2-7: GC-MS analysis of standard isobutanol and the Δcit3 strain. (A) Specific ion chromatograph (m/z 57) of standard isobutanol. (B) Mass spectra of standard isobutanol. (C) Specific ion chromatograph (m/z 57) of Δcit3 grown in YPD medium. (D) Mass spectra of isobutanol in the Δcit3 strain.

Table 2-1: List of primers used to generate CIT1 and CIT3 cDNAs.

Primer Sequence 5’ Æ 3’ Express Forward GAG GTA TAT ATA TTA ATG TAT CG CIT1 Forward ATG TCA GCG ATA TTA TCA A CIT1 Reverse TTA GTT CTT ACT TTC GAT TT CIT3 Forward ATG GTA CAA AGG CTT CTA C CIT3 Reverse TTA CAA CTT GTT AAC ATT GC

Table 2-2: Percent identity and similarity among citrate synthase homologues. The calculation of percent identity was obtained by dividing the total number of amino acids which are identical between each sequence by the number of amino acids of that sequence being compared to (Cit1p has 478 residues while Cit3p has 486 residues). The calculation of percent similarity was obtained by dividing the total number of amino acids which are identical plus any amino acid similar as defined in the Materials and Methods by the number of amino acids of that sequence being compared to. a, Sus scrofa. b, Gallus gallus. c, citrate synthase in E. coli. d, methylcitrate synthase in E. coli.

Cit1p Percent Identity Percent Similarity Cit3p 44.4 53.8 PIGa 55.4 65.3 CHICKb 53.6 63.0 GltAc 20.1 28.7 PrpCd 17.6 28.9

Cit3p Percent Identity Percent Similarity Cit1p 43.6 52.9 PIG 42.6 51.6 CHICK 39.9 48.8 GltA 20.2 26.8 PrpC 16.5 26.1

Table 2-3: Substrate specificities of recombinant Cit1p and Cit3p. Enzyme assay and determination of kinetic parameters were performed as described in Materials and Methods. a, no activity.

Cit1p Cit3p Acetyl-CoA Propionyl-CoA Acetyl-CoA Propionyl-CoA a Km (μM) 76 +/- 7 NA 1200 +/- 200 520 +/- 90 -1 kcat (s ) 11.2 +/- 0.4 NA 11 +/- 1 5.4 +/- 0.5 kcat / Km (x103 M-1 s-1) 150 +/- 10 NA 9 +/- 2 10 +/- 2

Table 2-4: Native citrate synthase and methylcitrate synthase activities of WT, Δcit1, and Δcit3. Sample preparation and enzyme assay were performed as described in Materials and Methods.

Acetyl-CoA Propionyl-CoA Acetyl-CoA / Media, Type (μmol/min/mg) (μmol/min/mg) Propionyl-CoA YPD, WT 0.177 +/- 0.007 0.0016 +/- 0.0004 110 +/- 20 YPD, Δcit1 1.7 +/- 0.3 0.21 +/- 0.04 8 +/- 2 YPD, Δcit3 0.15 +/- 0.02 0.0013 +/- 0.0002 120 +/- 20

YPG, WT 0.46 +/- 0.06 0.046 +/- 0.006 10 +/- 2 YPG, Δcit1 1.7 +/- 0.4 0.39 +/- 0.09 4 +/- 1 YPG, Δcit3 0.6 +/- 0.1 0.077 +/- 0.004 7 +/- 2

YPE, WT 0.340 +/- 0.01 0.0074 +/- 0.0005 46 +/- 4 YPE, Δcit1 1.02 +/- 0.07 0.39 +/- 0.01 2.6 +/- 0.2 YPE, Δcit3 0.45 +/- 0.07 0.0064 +/- 0.0003 70 +/- 10

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