Structural Characterization of the Molecular Events during a Slow Substrate-Product Transition in Orotidine 5′-Monophosphate Decarboxylase

JOURNAL OF MOLECULAR BIOLOGY

卷:387

Article

Fujihashi, Masahiro^1,2  Wei, Lianhu^3,4  Kotra, Lakshmi P.^3,4,5,6,7  Pai, Emil F.^2,8,9,10 

[1] Kyoto Univ, Grad Sch Sci, Sakyo Ku, Kyoto 6068502, Japan

[2] Toronto Med Discovery Tower, MaRS Ctr, Div Canc Genom & Proteom, Toronto, ON M5G 1L7, Canada

[3] Toronto Med Discovery Tower, MaRS Ctr, Toronto Gen Res Inst, Ctr Mol Design & Preformulat, Toronto, ON M5G 1L7, Canada

[4] Toronto Med Discovery Tower, MaRS Ctr, Toronto Gen Res Inst, Div Cell & Mol Biol, Toronto, ON M5G 1L7, Canada

[5] Univ Toronto, Dept Pharmaceut Sci, Toronto, ON, Canada

[6] Univ Toronto, Dept Chem, Toronto, ON M5S 1A1, Canada

[7] Univ N Carolina, Dept Chem & Biochem, Greensboro, NC 27402 USA

[8] Univ Toronto, Dept Biochem, Toronto, ON M5S 1A8, Canada

[9] Univ Toronto, Dept Med Biophys, Toronto, ON M5S 1A8, Canada

[10] Univ Toronto, Dept Mol Genet, Toronto, ON M5S 1A8, Canada

关键词: ODCase;    orotidine monophosphate decarboxylase;    OMPDC;    covalent inhibition;    time-resolved crystallography;   

DOI  :  10.1016/j.jmb.2009.02.037

来源: Elsevier

【 摘 要 】

Crystal structures of substrate-product complexes of Methanobacterium thermoautotrophicum orotidine 5'-monophosphate decarboxylase, obtained at various steps in its catalysis of the unusual transformation of 6-cyanouridine 5'-monophosphate (UMP) into barbituric acid ribosyl monophosphate, show that the cyano substituent of the substrate, when bound to the active site, is first bent significantly from the plane of the pyrimidine ring and then replaced by an oxygen atom. Although the K72A and D70A/K72A mutants are either catalytically impaired or even completely inactive, they still display bending of the C6 substituent. Interestingly, high-resolution structures of the D70A and D75N mutants revealed a covalent bond between C6 of UMP and the Lys72 side chain after the -CN moiety's release. The same covalent bond was observed when the native enzyme was incubated with 6-azido-UMP and 6-iodo-UMP; in contrast, the K72A mutant transformed 6-iodo-UMP to barbituric acid ribosyl 5'-monophosphate. These results demonstrate that, given a suitable environment, native orotidine 5'-monophosphate decarboxylase and several of its mutants are not restricted to the physiologically relevant decarboxylation; they are able to catalyze even nucleophilic substitution reactions but consistently maintain distortion on the C6 substituent as an important feature of catalysis. (C) 2009 Elsevier Ltd. All rights reserved.

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