Purification and characterization of 2-deoxy-scyllo-inosose synthase derived from Bacillus circulans. A crucial carbocyclization enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics.
The biosynthesis of 2-deoxystreptamine, the central aglycon of a major group of clinically important aminoglycoside antibiotics, commences with the initial carbocycle formation step from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. This crucial step is known to be catalyzed by 2-deoxy-scyllo-inosose synthase, which has not yet been characterized so far. Reported in this paper is the first purification of 2-deoxy-scyllo-inosose synthase from butirosin-producing Bacillus circulans SANK 72073 to electrophoretic homogeneity. The enzyme was isolated as a heterodimeric protein comprising from a 23 kDa- and a 42 kDa polypeptide chains. The Km of the enzyme for D-glucose-6-phosphate was estimated to be 9.0 x 10(-4) M and that for NAD+ 1.7 x 10(-4) M, kcat for D-glucose-6-phosphate being 7.3 x 10(-2) s(-1). The presence of Co2+ was essential for the enzyme activity, but Zn2+ was totally inhibitory. While the reaction mechanisms are quite similar, 2-deoxy-scyllo-inosose synthase appears to be distinct from dehydroquinate synthase in the shikimate pathway, with respect to the quaternary structure, metal ion requirement, and the kinetic parameters.
Kudo
F
F
Department of Chemistry, Tokyo Institute of Technology, Japan.
Hosomi
Y
Y
Tamegai
H
H
Kakinuma
K
K
D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D002076:Butirosin Sulfate / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D003500:Cyclization; D004591:Electrophoresis, Polyacrylamide Gel; D006595:Hexosamines / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet
D000900:Anti-Bacterial Agents; D006595:Hexosamines; D002076:Butirosin Sulfate; D008190:Lyases; C085631:2-deoxy-scyllo-inosose synthase; C007265:2-deoxystreptamine