Functional Analysis of a Gene Cluster from Involved in Biosynthesis of the Pyrrolidine Azasugar DAB-1.

Azasugars, "nitrogen in the ring" analogues of monosaccharides, are known to be distributed in select plant, fungal. and bacterial species. We identify DSM 2588 as the first bacterial source of the plant pyrrolidine azasugar 1,4-dideoxy-1,4-aminoarabinitol (DAB-1). Comparative sequence analyses identified as a putative azasugar producer, via observation of a three-gene cluster coding for putative aminotransferase, alcohol dehydrogenase, and sugar phosphatase enzymes, similar to the previously reported azasugar biosynthetic signature identified in FZB42. Multistep fractionation of culture media guided by a maltase inhibition assay yielded a component with a mass consistent with the structure of DAB-1. Heterologous expression of the three-gene cluster in , a non-azasugar producer, led to the isolation of nectrisine, a biosynthetic precursor to DAB-1, which displayed potent slow tight binding inhibition of maltase. Reduction of nectrisine with NaBH removed the slow tight binding inhibition kinetics, and MS analysis provided evidence for the production of a compound matching that of the isolated DAB-1 from . H NMR analysis of the nectrisine produced in after NaBD reduction produced a spectrum consistent with DAB-1 deuterated at C-1, primarily at the pro-S position. These results support the idea that the azasugar three-gene cluster represents a general biosynthetic path leading to several different compounds, which may prove useful for the identification of other azasugar-producing organisms.