TY - JOUR
T1 - Evolution of enzymatic activities in the orotidine 5′-monophosphate decarboxylase suprafamily
T2 - Mechanistic evidence for a proton relay system in the active site of 3-keto-L-gulonate 6-phosphate decarboxylase
AU - Yew, Wen Shan
AU - Wise, Eric L.
AU - Rayment, Ivan
AU - Gerlt, John A.
PY - 2004/6/1
Y1 - 2004/6/1
N2 - 3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5′-monophosphate decarboxylase (OMPDC) are homologous enzymes that share the (β/α)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 β-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 Mg2+-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 β-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 β-strand, their roles in catalysis are not conserved. Furthermore, a conserved Asp at the end of the third β-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.
AB - 3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5′-monophosphate decarboxylase (OMPDC) are homologous enzymes that share the (β/α)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 β-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 Mg2+-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 β-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 β-strand, their roles in catalysis are not conserved. Furthermore, a conserved Asp at the end of the third β-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.
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U2 - 10.1021/bi049741t
DO - 10.1021/bi049741t
M3 - Article
C2 - 15157077
AN - SCOPUS:2542531685
SN - 0006-2960
VL - 43
SP - 6427
EP - 6437
JO - Biochemistry
JF - Biochemistry
IS - 21
ER -