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.
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