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.
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.
Nango
Eriko
E
Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan.
Eguchi
Tadashi
T
Kakinuma
Katsumi
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006595:Hexosamines / Q000096:biosynthesis*; D008190:Lyases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D006595:Hexosamines; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008190:Lyases; C085631:2-deoxy-scyllo-inosose synthase; D005947:Glucose; D008239:Lysine; C007265:2-deoxystreptamine