d-Ribulose 5-Phosphate 3-Epimerase:  Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (β/α)₈-Barrel Superfamily^,

Abstract

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.

Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese

“…To study Rpe in vitro, Rpe from E. coli was purified using a His tag, which was removed after purification. Rpe is known to require a divalent metal for activity, but the identity of the metal used in vivo remained uncertain (25). A previous study of Rpe from Streptococcus pyogenes reported that it copurified with substantial Zn 2+ , leading the authors to speculate that this is its physiological metal.…”

“…A previous study of Rpe from Streptococcus pyogenes reported that it copurified with substantial Zn 2+ , leading the authors to speculate that this is its physiological metal. Akana et al (25) reported that Mn 2+ and Co 2+ also would activate Rpe in vitro, but Mg 2+ and Fe 2+ would not. However, these experiments were conducted under aerobic conditions, and the Fe 2+ probably oxidized to Fe 3+ .…”

“…coli Rpe was purified by His tag using a variation of the protocol described by Akana et al (25). Purification details are described in SI Materials and Methods.…”

“…Rpe interconverts ribulose-5-phosphate and xylulose-5-phosphate by abstracting a proton from one face of the C-3 carbon and then adding a proton to the opposite face. The metal coordinates substrate and stabilizes the intermediate oxyanion (25). (27), and the reduction of Fe by ascorbate regenerated Fe 2+ .…”

Structure of l -Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic l -Ascorbate Utilization Pathway of Escherichia coli : Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

“…The most similar (DALI [15] (1). RPE belongs to a TIM barrel family that is distinct from the family containing UlaE.…”