D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily.

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.

Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: enhancing the promiscuous D-arabino-hex-3-ulose 6-phosphate synthase reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase.

3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC) and d-arabino-hex-3-ulose 6-phosphate synthase (HPS) are members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869], a group of homologous enzymes that share the (beta/alpha)(8)-barrel fold. KGPDC catalyzes a Mg(2+)-dependent decarboxylation reaction 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]; HPS catalyzes a Mg(2+)-dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate in formaldehyde-fixing methylotrophic bacteria [Kato, N., Ohashi, H., Hori, T., Tani, Y., and Ogata, K. (1977) Agric. Biol. Chem. 41, 1133-1140]. Our previous studies of the KGPDC from E. coli established the occurrence of a stabilized cis-enediolate intermediate [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437; Wise, E., Yew, W. S., Gerlt, J. A., and Rayment, I. (2004) Biochemistry 43, 6438-6446]. Although the mechanism of the HPS-catalyzed reaction has not yet been investigated, it also is expected to involve a Mg(2+)-stabilized cis-enediolate intermediate. We now have discovered that the KGPDC from E. coli and the HPS from Methylomonas aminofaciens are both naturally promiscuous for the reaction catalyzed by the homologue. On the basis of the alignment of the sequences of orthologous KGPDC's and HPS's, four conserved active site residues in the KGPDC from E. coli were mutated to those conserved in HPS's (E112D/R139V/T169A/R192A): the value of the k(cat) for the promiscuous HPS activity was increased as much as 170-fold (for the E112D/R139V/T169A/R192A mutant), and the value of k(cat)/K(m) was increased as much as 260-fold (for the E112D/R139V/T169A mutant); in both cases, the values of the kinetic constants for the natural KGPDC activity were decreased. Together with the structures of mutants reported in the accompanying manuscript [Wise, E. L., Yew, W. S., Akana, J., Gerlt, J. A., and Rayment, I., accompanying manuscript], these studies illustrate that large changes in catalytic efficiency can be accomplished with only modest changes in active site structure. Thus, the (beta/alpha)(8)-barrel fold shared by members of the OMPDC suprafamily appears well-suited for the evolution of new functions.

Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: structural basis for catalytic promiscuity in wild-type and designed mutants of 3-keto-L-gulonate 6-phosphate decarboxylase.

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and D-arabino-hex-3-ulose 6-phosphate synthase (HPS), members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily, catalyze reactions that involve the formation of Mg(2+)-ion stabilized 1,2-enediolate intermediates. The active sites of KGPDC and HPS share several conserved residues, including the presumed ligands for the Mg(2+) and a catalytic histidine residue that has been implicated in protonation of the intermediate in the KGPDC-catalyzed reaction. As reported in the previous manuscript, both enzymes are naturally promiscuous, with KGPDC from Escherichia coli catalyzing a low level of the HPS reaction and the HPS from Methylomonas aminofaciens catalyzing a significant level of the KGPDC reaction. Interestingly, the promiscuous HPS reaction catalyzed by KGPDC can be significantly enhanced by replacing no more than four active site residues from KGPDC reaction with residues from HPS. In this manuscript, we report structural studies of wild-type and mutant KDGPC's that provide a structural explanation for both the natural promiscuity for the HPS reaction and the enhanced HPS activity and diminished KGPDC activity catalyzed by active site mutants.

Structure of D-ribulose 5-phosphate 3-epimerase from Synechocystis to 1.6 A resolution.

The crystal structure of D-ribulose 5-phosphate 3-epimerase (RPE) from the cyanobacterium Synechocystis was determined by X-ray crystallography to 1.6 A resolution. The enzyme, which catalyzes the epimerization of D-ribulose 5-phosphate and D-xylulose 5-phosphate, assembles as a hexamer of (beta/alpha)(8)-barrels in the crystallographic asymmetric unit. The active site is highly similar to those of two previously reported RPEs and provides further evidence for essential catalytic roles for several active-site residues.