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
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
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DOI：
10.1021/jo034706y
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发表时间：
2004-02-06
影响因子：
3.6
通讯作者：
Kakinuma, K
中科院分区：
文献类型：
--
作者：
Nango, E;Eguchi, T;Kakinuma, K
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 alpha,beta-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 alpha,beta-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.
