Evolution of Enzymatic Activities in the OMPDC Suprafamily: Discovery, Design and Evolution of 3-Keto-L-Gulonate 6-Phosphate Decarboxylase
Yew, Wen Shan
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https://hdl.handle.net/2142/84808
Description
Title
Evolution of Enzymatic Activities in the OMPDC Suprafamily: Discovery, Design and Evolution of 3-Keto-L-Gulonate 6-Phosphate Decarboxylase
Author(s)
Yew, Wen Shan
Issue Date
2004
Doctoral Committee Chair(s)
Gerlt, John A.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) was discovered to be involved in the fermentative utilization of L-ascorbate by enteric bacteria such as E. coli K-12. KGPDC is a new member of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily, and its discovery expands the range of reactions catalyzed by members of the suprafamily beyond non-cofactor-assisted decarboxylation (avoidance of a vinyl anion intermediate in the context of OMPDC), aldol-condensation (through divalent metal-ion stabilization of an enediolate intermediate in the context of HPS), and metal-independent epimerization (through active site stabilization of an enediolate intermediate in the context of RPE). The mechanism of the KGPDC-catalyzed reaction was determined, and it involves Mg2+-stabilization of an enediolate intermediate and a unique proton-relay system that provides the proton source for catalysis. KGPDC, like members of the OMPDC suprafamily, are dimers of (beta/alpha)8-barrels; the active site is located at the C-terminal ends of the beta-strands and comprises of residues originating from both monomers. The positions of functional groups are conserved amongst suprafamily members, although active site residues differ both in identity and mechanistic utility: in OMPDC, a conserved Lys at the ends of the 2nd beta-strand is involved in a charge-relay network that is required for catalysis; in KGPDC, a conserved Glu at the same position is recruited to stabilize a Mg 2+-ion that is required for stabilization of the ensuing enediolate intermediate during catalysis. By rational design, the promiscuity of the KGPDC scaffold towards catalysis of the HPS reaction was enhanced: three substitutions of E112D, R139V and T169A resulted in a 260-fold increase in catalytic efficiency, while the physiological KGPDC activity was decreased by 30-fold. These studies provided an initial insight into the opportunistic ways of Nature in bridging between points along the evolutionary landscape through the use of an active site architecture that is capable of catalyzing a reaction that has neither a semblance of chemical mechanism nor binding specificity to that of the progenitorial reaction.
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