Biosynthesis of the azasugar 1-deoxynojirimycin (DNJ) critically involves a transamination in the first committed step. Here, we identify the azasugar biosynthetic cluster signature in Paenibacillus polymyxa SC2 (Ppo), homologous to that reported in Bacillus amyloliquefaciens FZB42 (Bam), and report the characterization of the aminotransferase GabT1 (named from Bam). GabT1 from Ppo exhibits a specific activity of 4.9 nmol/min/mg at 30°C (pH 7.5), a somewhat promiscuous amino donor selectivity, and curvilinear steady-state kinetics that do not reflect the predicted ping-pong behavior typical of aminotransferases. Analysis of the first half reaction with l-glutamate in the absence of the acceptor fructose 6-phosphate revealed that it was capable of catalyzing multiple turnovers of glutamate. Kinetic modeling of steady-state initial velocity data was consistent with a novel hybrid branching kinetic mechanism which included dissociation of PMP after the first half reaction to generate the apoenzyme which could bind PLP for another catalytic deamination event. Based on comparative sequence analyses, we identified an uncommon His-Val dyad in the PLP-binding pocket which we hypothesized was responsible for the unusual kinetics. Restoration of the conserved PLP-binding site motif via the mutant H119F restored classic ping-pong kinetic behavior.

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