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

摘要

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 coll 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 His 136 and Argl39,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 Ol 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.