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MIAMI UNIVERSITY the Graduate School Certificate for Approving The MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Matthew F. Rouhier Candidate for the Degree: Doctor of Philosophy ___________________________________________________________ Director (Dr. Ann Hagerman) ___________________________________________________________ Reader (Dr.Chris Makaroff) ___________________________________________________________ Reader (Dr.Gary Lorigan) __________________________________________________________ Reader (Dr. Richard Taylor) _________________________________________________________ Graduate School Representative (Dr. Paul James) ! ABSTRACT CHARACTERIZATION OF YDR036C FROM Saccharomyces cerevisiae by Matthew F. Rouhier Beta-hydroxyisobutyryl-CoA (HIBYL-CoA) hydrolases are found ubiquitously in eukaryotes where they function in the catabolism of valine. A homologous enzyme (YDR036C) is also present in yeast where valine catabolism is distinctly different and does not require a HIBYL-CoA hydrolase. Like the other eukaryotic hydrolases, the yeast hydrolase is a member of the crotonase super-family which catalyzes various reactions using CoA thioester substrates. Crotonases typically function in fatty acid degradation which is peroxisomal in yeast while YDR036C is found strictly in mitochondria. Like other HIBYL-CoA hydrolases the yeast enzyme demonstrated activity toward beta-hydroxyacyl-CoAs, but unlike the other HIBYL-CoA hydrolases the yeast activity is greatest with beta-hydroxypropionyl-CoA (3-HP-CoA). Further characterization of the active site has determined that the preference of the hydrolases for 3-HP- CoA or HIBYL-CoA is dependent upon the residue at position 177. The glutamate at position 121 is responsible for the coordination with the beta-hydroxyl group and replacement with valine renders the enzyme non-specific for a hydroxyl group. Another unique feature to yeast hydrolases is the presence of a C-terminal tail of 80 amino acids, which when removed renders the enzyme inactive, suggesting that it may play a role in substrate binding. Serial truncation of the enzyme demonstrated that the tail residues between 472 and 500 are critical for the proper hydrolase activity. Similarly the enzyme can be inactivated by placing a phosphate ester-mimic in place of serine 428. Although the active site seems optimized for 3-HP-CoA this compound is not a known metabolite of yeast. This suggests that YDR036C may be serving a unique function in yeast. High-throughput studies have identified a mild phenotype in fluid-phase endoyctosis in ydr036c mutants. The same phenotype is observed in knockouts of ergosterol synthesis enzymes. Ergosterol quantification in the ydr036c knockout showed reduced total cellular ergosterol when compared to wild-type. Ergosterol was subsequently docked to a model of the enzyme and shown to bind at the active site pocket. Since the storage mechanism for yeast sterols is sterol esters, this study proposes that YDR036C is not a HIBYL-CoA hydrolase, but instead may function as a mitochondrial sterol esterase. ! CHARACTERIZATION OF YDR036C FROM Saccharomyces cerevisiae A DISSERTATION Submitted to the Faculty of Miami University in partial Fulfillment of the requirements for a degree of Doctor of Philosophy Department of Chemistry and Biochemistry by Matthew F. Rouhier Miami University Oxford, Ohio 2011 ! Table of Contents Chapter 1: Introduction ............................................................................................................. 1 1.1 Crotonase ......................................................................................................................... 1 1.1.1 Crotonase ................................................................................................................. 1 1.2 !-hydroxyisobutyryl-CoA hydrolase ............................................................................... 1 1.2.1 HIBYL-CoA hydrolase homology .......................................................................... 1 1.2.2 HIBYL-CoA hydrolase and valine metabolism ...................................................... 2 1.2.3 HIBYL-CoA hydrolase structure ............................................................................ 2 1.2.4 HIBYL-CoA hydrolase mechanism ........................................................................ 2 1.2.5 HIBYL-CoA hydrolase specificity .......................................................................... 2 1.3 References ....................................................................................................................... 4 Chapter 2: Characterization of YDR036C .............................................................................. 10 2.1 Introduction ................................................................................................................... 10 2.1.1 YDR036C and HIBYL-CoA hydrolase ................................................................. 10 2.2 Materials and Methods .................................................................................................. 11 2.2.1 Materials and cell lines .......................................................................................... 11 2.2.2 Cloning and mutagenesis ....................................................................................... 11 2.2.3 Protein over-expression and purification ............................................................... 12 2.2.4 Assay of hydrolase activity ................................................................................... 13 2.2.5 pH profile of YDR036C and variants .................................................................... 13 2.2.6 Modeling of YDR036C ......................................................................................... 13 2.2.7 Docking of substrates ............................................................................................ 13 2.3 Results ........................................................................................................................... 14 2.3.1 Sequence alignment & homology ......................................................................... 14 2.3.2 Hydrolase activities ............................................................................................... 14 ""!! 2.3.3 Modeling and docking ........................................................................................... 15 2.3.4 Residues controlling methyl group preference ...................................................... 15 2.3.5 Residues controlling hydroxyl group and carbon chain requirement .................... 16 2.4 Discussion ...................................................................................................................... 17 2.5 References ..................................................................................................................... 19 Chapter 3: Phosphorylation of HIBYL-CoA hydrolase .......................................................... 29 3.1 Introduction ................................................................................................................... 29 3.1.1 HIBYL-CoA hydrolase regulation ........................................................................ 29 3.1.2 Mitochondrial regulation by phosphorylation ....................................................... 29 3.1.3 Determination of phosphorylation state ................................................................ 29 3.2 Materials and Methods .................................................................................................. 29 3.2.1 Cloning and mutagenesis ....................................................................................... 29 3.2.2 Assay of hydrolase activity ................................................................................... 30 3.3 Results and Discussion .................................................................................................. 30 3.3.1 Sequence alignment of HIBYL-CoA hydrolases .................................................. 30 3.3.2 Phosphorylation mimics ........................................................................................ 30 3.3.3 Future work ........................................................................................................... 31 3.4 References ..................................................................................................................... 32 Chapter 4: Potential role of YDR036C in regulating ergosterol metabolism ......................... 38 4.1 Introduction ................................................................................................................... 38 4.1.1 YDR036C homology to valine hydrolase ............................................................. 38 4.1.2 Hydrolase activity .................................................................................................. 38 4.1.3 Homology modeling .............................................................................................. 38 4.1.4 Ergosterol .............................................................................................................. 39 4.1.5 Synthesis of ergosterol in the ER .......................................................................... 39 """!! 4.1.6 Shuttling of ergosterol to the LP ........................................................................... 40 4.1.7 Shuttling of ergosterol to the PM .......................................................................... 40 4.1.8 Detoxification
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