Publications

• Structural evidence for a 1,2-enediolate intermediate in the reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase, a member of the orotidine 5'-monophosphate decarboxylase suprafamily.

  Wise E.L., Yew W.S., Gerlt J.A., Rayment I.

  3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5'-phosphate decarboxylase (OMPDC) are members of an enzyme suprafamily, the OMPDC suprafamily, because they are homologous enzymes that catalyze mechanistically distinct reactions using different substrates. KGPDC catalyzes the Mg( ... >> More

  3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5'-phosphate decarboxylase (OMPDC) are members of an enzyme suprafamily, the OMPDC suprafamily, because they are homologous enzymes that catalyze mechanistically distinct reactions using different substrates. KGPDC catalyzes the Mg(2+) ion-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate to yield L-xylulose 5-phosphate and CO(2); OMPDC catalyzes the metal ion-independent decarboxylation of OMP to UMP and CO(2). Structural studies have shown that KGPDC and OMPDC share several strictly conserved active site residues that are used differently by each enzyme to catalyze their mechanistically distinct reactions. Although the mechanism of the KGPDC-catalyzed reaction has yet to be elucidated, it is thought to proceed via a Mg(2+) ion-stabilized 1,2-enediolate intermediate. Here we report the crystal structures of KGPDC complexed with L-gulonate 6-phosphate, L-threonohydroxamate 4-phosphate, and L-xylitol 5-phosphate, analogues of the substrate, enediolate intermediate, and product, as well as with the product, L-xylulose 5-phosphate, at 1.2, 1.8, 1.7, and 1.8 A resolution, respectively. These structures support a mechanism that involves the formation of a cis-1,2-enediolate intermediate. Contrary to expectations, the geometry of the intermediate does not involve bidentate coordination of both enediolate oxygen atoms to the Mg(2+) ion but rather involves only the coordination of the oxygen on C2 to the Mg(2+) ion. The oxygen atom on C1 instead forms hydrogen bonds to both Lys64 and Asp67, two strictly conserved active site residues. Lys64 also interacts with the oxygen on C2 and may serve to stabilize a cis conformation of the 1,2-enediolate. These structures also implicate His136 to be the general acid that protonates the 1,2-enediolate intermediate. This study further demonstrates that multiple unrelated enzyme functions can evolve from a single active site architecture without regard for substrate binding affinity or mechanism. << Less

  Biochemistry 42:12133-12142(2003) [PubMed] [EuropePMC]

• Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: mechanistic evidence for a proton relay system in the active site of 3-keto-L-gulonate 6-phosphate decarboxylase.

  Yew W.S., Wise E.L., Rayment I., Gerlt J.A.

  3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5'-monophosphate decarboxylase (OMPDC) are homologous enzymes that share the (beta/alpha)(8)-fold but catalyze mechanistically distinct reactions [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemist ... >> More

  3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5'-monophosphate decarboxylase (OMPDC) are homologous enzymes that share the (beta/alpha)(8)-fold but catalyze mechanistically distinct reactions [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869]. KGPDC catalyzes the Mg(2+)-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate, an intermediate in the catabolic pathway of L-ascorbate utilization by Escherichia coli K-12 [Yew, W. S., and Gerlt, J. A. (2002) J. Bacteriol. 184, 302-306]. OMPDC catalyzes a metal ion-independent reaction that likely proceeds without a vinyl anion intermediate [Appleby, T. C., Kinsland, C., Begley, T., and Ealick, S. E. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 2005-2010], although the mechanistic details are uncertain. An active site Lys located at the end of the third beta-strand in OMPDC has been proposed to be the general acid that delivers a solvent-derived proton to the UMP product; the active site of KGPDC contains a homologous Lys residue (Lys64). Herein, we report investigations of the KGPDC-catalyzed reaction that are consistent with a mechanism involving a Mg(2+)-stabilized cis-enediolate intermediate [Wise, E. L., Yew, W. S., Gerlt, J. A., and Rayment, I. (2003) Biochemistry 42, 12133-12142] and implicate waters proximal to His136 and Arg139, both located at the end of the sixth beta-strand, as the general acids that deliver a solvent-derived proton to the intermediate to form the L-xylulose 5-phosphate product. On the basis of our mechanistic investigations, Lys64 stabilizes the cis-enediolate intermediate by forming hydrogen bonds to both O1 and O2 of the intermediate. Thus, although the active sites of OMPDC and KGPDC contain a conserved Lys at the end of the third beta-strand, their roles in catalysis are not conserved. Furthermore, a conserved Asp at the end of the third beta-strand in OMPDC participates in a hydrogen-bonded network that positions the acidic Lys residue; in the active site of KGPDC, the homologous Asp67 participates in stabilization of the enediolate intermediate and enforces a cis geometry. We conclude that the conserved active site residues perform different functions in the OMPDC- and KGPDC-catalyzed reactions, so the mechanisms of their reactions are completely distinct. This study further highlights the opportunistic nature of divergent evolution in conscripting the active site of a progenitor to catalyze a mechanistically distinct reaction. << Less

  Biochemistry 43:6427-6437(2004) [PubMed] [EuropePMC]

• Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: crystallographic evidence for a proton relay system in the active site of 3-keto-L-gulonate 6-phosphate decarboxylase.

  Wise E.L., Yew W.S., Gerlt J.A., Rayment I.

  3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC), a member of the orotidine monophosphate decarboxylase (OMPDC) suprafamily, catalyzes the Mg(2+)-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate to L-xylulose 5-phosphate. Structural and biochemical evidence suggests that the KGPDC rea ... >> More

  3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC), a member of the orotidine monophosphate decarboxylase (OMPDC) suprafamily, catalyzes the Mg(2+)-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate to L-xylulose 5-phosphate. Structural and biochemical evidence suggests that the KGPDC reaction proceeds via a Mg(2+)-stabilized 1,2-cis-enediolate intermediate. Protonation of the enediolate intermediate occurs in a nonstereospecific manner to form L-xylulose 5-phosphate. Although the exact mechanism of proton delivery is not known, Glu112, His136, and Arg139 have been implicated in this process [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437]. Surprisingly, single amino acid substitutions of these positions do not substantially reduce catalytic activity but rather alter the stereochemical course of the reaction. Here, we report the X-ray crystal structures of four mutants, K64A, H136A, E112Q, and E112Q/H136A, each determined in the presence of L-threonohydroxamate 4-phosphate, an analogue of the enediolate intermediate, to 1.7, 1.9, 1.8, and 1.9 A resolution, respectively. These structures reveal that substitutions of Lys64, Glu112, and His136 cause changes in the positions of the intermediate analogue and two active site water molecules that were previously identified as possible proton donors. These changes correlate with the observed alterations in the reaction stereochemistry for these mutants, thereby supporting a reaction mechanism in which water molecules competitively shuttle protons from the side chains of His136 and Arg139 to alternate faces of the cis-enediolate intermediate. These studies further underscore the wide variation in the reaction mechanisms in the OMPDC suprafamily. << Less

  Biochemistry 43:6438-6446(2004) [PubMed] [EuropePMC]

• Homologous (beta/alpha)8-barrel enzymes that catalyze unrelated reactions: orotidine 5'-monophosphate decarboxylase and 3-keto-L-gulonate 6-phosphate decarboxylase.

  Wise E., Yew W.S., Babbitt P.C., Gerlt J.A., Rayment I.

  The 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC) encoded by the ulaD gene in the Escherichia coli genome [Yew, W. S., and Gerlt, J. A. (2002) J. Bacteriol. 184, 302-306] and orotidine 5'-monophosphate decarboxylase (OMPDC) are homologous (derived from a common ancestor) but catalyze differe ... >> More

  The 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC) encoded by the ulaD gene in the Escherichia coli genome [Yew, W. S., and Gerlt, J. A. (2002) J. Bacteriol. 184, 302-306] and orotidine 5'-monophosphate decarboxylase (OMPDC) are homologous (derived from a common ancestor) but catalyze different reactions. The metal-independent decarboxylation reaction catalyzed by OMPDC avoids the formation of a vinyl anion intermediate; the Mg2+-dependent decarboxylation reaction catalyzed by KGPDC involves the formation of an enediolate anion intermediate. Based on the available structures of OMPDC, a sequence alignment allows the predictions that (1) KGPDC is a dimer of (beta/alpha)8-barrels, with the active sites located at the dimer interface; (2) KGPDC and OMPDC share an aspartate residue at the end of the first beta-strand and an Asp-x-Lys-x-x-Asp motif at the end of the third beta-strand with OMPDC; but (3) KGPDC has a Glu instead of a Lys at the end of the second beta-strand. The structure of KGPDC has been determined in the presence of Mg2+ and the substrate analogue L-gulonate 6-phosphate and confirms these predictions. The carboxylate functional groups at the ends of the first, second, and third beta-strands in KGPDC are ligands of the Mg2+; in OMPDC, the homologues of these residues participate in a hydrogen-bonded network that facilitates the decarboxylation reaction. The 3-OH group of the substrate analogue is coordinated to the Mg2+, supporting the hypothesis that the mechanism of the decarboxylation catalyzed by KGPDC involves stabilization of an enediolate anion intermediate. These structural studies establish the existence of the OMPDC "suprafamily," in which members catalyze reactions that occur in different metabolic pathways and share no mechanistic relationship. The existence of this suprafamily demonstrates that divergent evolution can be opportunistic, conscripting active site features of a progenitor to catalyze unrelated functions. Accordingly, sequence or structure homology alone cannot be used to infer the functions of new proteins discovered in genome projects. << Less

  Biochemistry 41:3861-3869(2002) [PubMed] [EuropePMC]