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.
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.
Yew
Wen Shan
WS
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Wise
Eric L
EL
Rayment
Ivan
I
Gerlt
John A
JA
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D005557:Formaldehyde / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry* / Q000378:metabolism*; D010428:Pentosephosphates / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry; D013237:Stereoisomerism
D029968:Escherichia coli Proteins; D010428:Pentosephosphates; D011522:Protons; D012997:Solvents; D005557:Formaldehyde; D006639:Histidine; C031625:xylulose-5-phosphate; D001120:Arginine; C451737:3-keto-L-gulonate 6-phosphate decarboxylase, E coli; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase; D008239:Lysine