Azasugars are so-called �nitrogen in the ring� analogs of the common monosaccharides in which the endocyclic ring oxygen is replaced by nitrogen. The archetypical examples in the class are the nojirimycins, well known glycosidase inhibitors produced by microbial and plant species. We defined a three gene cluster consisting of transaminase, phosphatase and dehydrogenase activities as sufficient for production of mannojirimycin in Bacillus. The biosynthetic pathway begins with amination of fructose-6-phosphate by a PLP-dependant transaminase termed GabT1 in Bacillus amyloliquefaciens. Here we wish to describe characterization of a homologous enzyme in Paenibacillus polymyxa, ppsc2_c2585, which is found in an azasugar signature cluster. This enzyme is a member of the OAT family of aminotransferases. The codon optimized gene was expressed in E. coli BL21(DE3) (4 mg/L) and purified by immobilized metal affinity chromatography. Two different coupled assays for the enzymes were developed, both based on detection of NADH. The first uses alpha ketoglutarate dehydrogenase to detect alpha ketoglutarate, generated by deamination of the amino donor substrate glutamate. The other assay uses aminomannitol dehydrogenase (AMDH) which is also part of the azasugar biosynthetic pathway. Using these assays we determined that fructose or fructose-6-phosphate can function as an amino acceptor, though fructose-6-phosphate is preferred. The specific activity for the P . polymyxa enzyme is 0.4 nmol/min/mg with 2 mM glutamate and fructose, while with fructose-6-phosphate as an amino acceptor the specific activity is 1 nmol/min/mg (pH 7.5, 30 �C). This enzyme is distinct from glutamine-fructose-6-phosphate transaminase (isomerizing, EC 2.6.1.16) because it is a PLP dependant enzyme whose product is 2-aminomannitol-6-phosphate.

Characterization of the PLP dependent transaminase initiating azasugar synthesis

248th National Meeting (2014)

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