Divergent Evolution of Enzymes in the Crotonase Superfamily: Exploring Functional Diversity and Challenging Mechanistic Paradigms
Wong, Brian James
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https://hdl.handle.net/2142/84142
Description
Title
Divergent Evolution of Enzymes in the Crotonase Superfamily: Exploring Functional Diversity and Challenging Mechanistic Paradigms
Author(s)
Wong, Brian James
Issue Date
2004
Doctoral Committee Chair(s)
Gerlt, John A.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
The study of enzyme superfamilies is an approach used to solve problems in the field of genomic enzymology. Research on the crotonase superfamily seeks to accomplish three goals. The first is to use homology searches to predict and assign functions to unknown proteins in sequenced databases. The second is to use the concept of the enzyme superfamily to elucidate structure-function relationships within the protein itself. The final goal is to be able to use this mechanistic information to gain insight into the evolution of catalytic activity. BLAST searches, sequence alignments, and metabolic contexts were used to locate and identify two unknown proteins in the genome of Pseudomonas species. The proteins were putatively identified as 3-methylglutaconyl-CoA hydratase (MGCH)---an enzyme in leucine catabolism, and 3-hydroxyisobutyryl-CoA hydrolase (HICH)---an enzyme involved in valine catabolism. Studies of the mechanism of MGCH showed that the enzyme catalyzed the hydration of methylglutaconyl-CoA to (3S)-hydroxymethylgluatryl-CoA. Site-directed mutagenesis experiments showed that a single active-site residue (Glu 138) catalyzes this hydration. However, based on substrate specificity experiments, the possibility of an additional functional group---namely, the gamma-carboxylate of the substrate---carrying out this catalysis cannot be ruled out. HICH was shown to catalyze the hydrolysis of a thioester bond to yield hydroxyisobutyryl-CoA. It was shown the mechanism of HICH did not proceed via alpha-proton abstraction as one would normally predict for a member of the crotonase superfamily. Rather, the mechanism involves the nucleophilic attack by an active-site carboxylate to form an anhydride intermediate which is subsequently hydrolyzed at the enzyme carbonyl carbon to give the product. Mutagenesis experiments revealed Glu 143 as the nucleophile in the reaction. Studies of these enzymes have demonstrated the versatility of conserved mechanistic and structural features within the superfamily. Nature has retained a single glutamate as an active-site residue but has utilized it in different roles in the two reactions. Similarly, the oxyanion hole is a structural feature conserved in all superfamily members; yet it stabilizes a different anionic intermediate in each of the two reactions.
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