D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (
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- 作者：Julie Akana ; Alexander A. Fedorov ; Elena Fedorov ; Walter R. P. Novak ; Patricia C. Babbitt ; Steven C. Almo ; and John A. Gerlt
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：February 28, 2006
- 年：2006
- 卷：45
- 期：8
- 页码：2493 - 2503
- 全文大小：514K
- 年卷期：v.45,no.8(February 28, 2006)
- ISSN：1520-4995
文摘
The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins(SCOP) database is considered the result of divergent evolution from a common (
/
)8-barrel ancestor.The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphatedecarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of theOMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilizephosphorylated metabolites as substrates. We now report studies of the functional and structural relationshipsof RPE to the members of the superfamily. As suggested by the results of crystallographic studies of theRPEs 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 activatedby Zn2+ which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a highaffinity for Zn2+ and inactive apoenzyme cannot be prepared, the affinity for Zn2+ is decreased by alaninesubstitutions for the two histidine residues that coordinate the Zn2+ ion (H34A and H67A); these mutantproteins can be prepared in an inactive, metal-free form and activated by exogenous Zn2+. The crystalstructure of the RPE was solved at 1.8 Å resolution in the presence of D-xylitol 5-phosphate, an inertanalogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediatein the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn2+ that also is ligandedto His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned alsoto function as the acid/base catalysts. Although the conformation of the bound analogue resembles thoseof ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues thatcoordinate the essential Zn2+ and participate as acid/base catalysts are not conserved. We conclude thatonly the phosphate binding motif located at the ends of the seventh and eighth -strands of the (/)8-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis thatthe members of the "ribulose phosphate binding" (/)8-barrel "superfamily" as defined by SCOP havenot evolved by evolutionary processes involving the intact (/)8-barrel. Instead, this "superfamily" mayresult from assembly from smaller modules, including the conserved phosphate binding motif associatedwith the C-terminal (/)2-quarter barrel.
/)8-barrel ancestor.The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphatedecarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of theOMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilizephosphorylated metabolites as substrates. We now report studies of the functional and structural relationshipsof RPE to the members of the superfamily. As suggested by the results of crystallographic studies of theRPEs 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 activatedby Zn2+ which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a highaffinity for Zn2+ and inactive apoenzyme cannot be prepared, the affinity for Zn2+ is decreased by alaninesubstitutions for the two histidine residues that coordinate the Zn2+ ion (H34A and H67A); these mutantproteins can be prepared in an inactive, metal-free form and activated by exogenous Zn2+. The crystalstructure of the RPE was solved at 1.8 Å resolution in the presence of D-xylitol 5-phosphate, an inertanalogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediatein the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn2+ that also is ligandedto His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned alsoto function as the acid/base catalysts. Although the conformation of the bound analogue resembles thoseof ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues thatcoordinate the essential Zn2+ and participate as acid/base catalysts are not conserved. We conclude thatonly the phosphate binding motif located at the ends of the seventh and eighth -strands of the (/)8-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis thatthe members of the "ribulose phosphate binding" (/)8-barrel "superfamily" as defined by SCOP havenot evolved by evolutionary processes involving the intact (/)8-barrel. Instead, this "superfamily" mayresult from assembly from smaller modules, including the conserved phosphate binding motif associatedwith the C-terminal (/)2-quarter barrel.