The kanosamine biosynthetic pathway in Bacillus cereus UW85: Functional and kinetic characterization of KabA, KabB, and KabC.

Kanosamine is an aminosugar antibiotic, and component of complex antibiotics such as kanamycin. The biosynthesis of kanosamine varies among different bacteria; best known is a pathway starting from UDP-glucose, but Bacillus subtilis can produce kanosamine in a three-step pathway from glucose 6-phosphate. A set of genes proposed to encode a kanosamine pathway has previously been identified within the zwittermicin A gene cluster of Bacillus cereus UW85. These genes, designated kabABC, are similar to the B. subtilis kanosamine pathway genes (ntdABC), but have never been studied experimentally. We have expressed each of the kab genes, and studied the in vitro substrate scope and reaction rates and kinetic mechanisms of all three enzymes. The kab genes encode enzymes catalyze a route similar to that found in B. subtilis from glucose 6-phosphate to kanosamine, passing through an unusual and unstable 3-keto intermediate. Kinetic studies show the first step in the pathway, the KabC-catalyzed oxidation of glucose 6-phosphate at carbon-3, is very slow relative to the subsequent KabA-catalyzed aminotransferase and KabB-catalyzed phosphatase reactions. KabC differs from its homolog, NtdC, in that it is NADP- rather than NAD-dependent. The KabA kinetic study is the first such report for a kanosamine 6-phosphate aminotransferase, revealing an extremely efficient PLP-dependent reaction. These results show that this kanosamine biosynthesis pathway occurs in more than one organism, and that the reactions are tuned in order to avoid any accumulation of the unstable intermediate.