Active Site Mapping of 2-Deoxy-scyllo-inosose Synthase, the Key Starter Enzyme for the Biosynthesis of 2-Deoxystreptamine. Mechanism-Based Inhibition and Identification of Lysine-141 As the Entrapped Nucleophile

Eriko Nango, Tadashi Eguchi, Katsumi Kakinuma

研究成果: Article査読

26 被引用数 (Scopus)

抄録

A key enzyme in the biosynthesis of clinically important aminoglycoside antibiotics including neomycin, kanamycin, gentamicin, etc. is 2-deoxy-scyllo-inosose synthase (DOIS), which catalyzes the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose (DOI). To clarify its precise reaction mechanism and crucial amino acid residues in the active site, we took advantage of a mechanism-based inhibitor carbaglucose-6-phosphate (pseudo-DL-glucose, C-6-P) with anticipation of its conversion to a reactive α,β-unsaturated carbonyl intermediate. It turned out that C-6-P clearly showed time- and concentration-dependent inhibition against DOIS, and the molecular mass of the resulting modified-DOIS with C-6-P was 160 mass units larger than that of native DOIS. Thus, the expected α,β-unsaturated intermediate appeared to trap a specific nucleophilic group in the active site through the Michael-type 1,4-addition. The covalently modified amino acid residue was determined to be Lys-141 by means of enzymatic digestion and subsequent LC/MS and LC/MS/MS of the digest. Also discussed are the role of Lys-141 in the substrate recognition and the reaction pathway and comparison with evolutionary related dehydroquinate synthase.

本文言語English
ページ(範囲)593-600
ページ数8
ジャーナルJournal of Organic Chemistry
69
3
DOI
出版ステータスPublished - 2004 2月 6
外部発表はい

ASJC Scopus subject areas

  • 有機化学

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