Evolution of Enzymatic Activities in the Orotidine 5'-Monophosphate Decarboxylase Suprafamily: Enhancing the Promiscuous D-arabino-Hex-3-ulose 6-Phosphate Synthase Reacti

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• 作者：Wen Shan Yew ; Julie Akana ; Eric L. Wise ; Ivan Rayment ; and John A. Gerlt
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：February 15, 2005
• 年：2005
• 卷：44
• 期：6
• 页码：1807 - 1815
• 全文大小：338K
• 年卷期：v.44,no.6(February 15, 2005)
• ISSN：1520-4995

文摘

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and D-arabino-hex-3-ulose 6-phosphatesynthase (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 (ges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">/ges/gifchars/alpha.gif" BORDER=0>)₈-barrel fold. KGPDC catalyzes a Mg²⁺-dependentdecarboxylation 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 Mg²⁺-dependent aldolcondensation between formaldehyde and D-ribulose 5-phosphate in formaldehyde-fixing methylotrophicbacteria [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 expectedto involve a Mg²⁺-stabilized cis-enediolate intermediate. We now have discovered that the KGPDC fromE. coli and the HPS from Methylomonas aminofaciens are both naturally promiscuous for the reactioncatalyzed by the homologue. On the basis of the alignment of the sequences of orthologous KGPDC'sand HPS's, four conserved active site residues in the KGPDC from E. coli were mutated to those conservedin HPS's (E112D/R139V/T169A/R192A): the value of the k_cat for the promiscuous HPS activity wasincreased as much as 170-fold (for the E112D/R139V/T169A/R192A mutant), and the value of k_cat/K_mwas increased as much as 260-fold (for the E112D/R139V/T169A mutant); in both cases, the values ofthe kinetic constants for the natural KGPDC activity were decreased. Together with the structures ofmutants reported in the accompanying manuscript [Wise, E. L., Yew, W. S., Akana, J., Gerlt, J. A., andRayment, I., accompanying manuscript], these studies illustrate that large changes in catalytic efficiencycan be accomplished with only modest changes in active site structure. Thus, the (ges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">/ges/gifchars/alpha.gif" BORDER=0>)₈-barrel fold sharedby members of the OMPDC suprafamily appears well-suited for the evolution of new functions.