The Biosynthesis of UDP-d-QuiNAc in Bacillus cereus ATCC 14579

N-acetylquinovosamine (2-acetamido-2,6-di-deoxy-d-glucose, QuiNAc) is a relatively rare amino sugar residue found in glycans of few pathogenic gram-negative bacteria where it can play a role in infection. However, little is known about QuiNAc-related polysaccharides in gram-positive bacteria. In a routine screen for bacillus glycan grown at defined medium, it was surprising to identify a QuiNAc residue in polysaccharides isolated from this gram-positive bacterium. To gain insight into the biosynthesis of these glycans, we report the identification of an operon in Bacillus cereus ATCC 14579 that contains two genes encoding activities not previously described in gram-positive bacteria. One gene encodes a UDP-N-acetylglucosamine C4,6-dehydratase, (abbreviated Pdeg) that converts UDP-GlcNAc to UDP-4-keto-4,6-d-deoxy-GlcNAc (UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose); and the second encodes a UDP-4-reductase (abbr. Preq) that converts UDP-4-keto-4,6-d-deoxy-GlcNAc to UDP-N-acetyl-quinovosamine in the presence of NADPH. Biochemical studies established that the sequential Pdeg and Preq reaction product is UDP-d-QuiNAc as determined by mass spectrometry and one- and two-dimensional NMR experiments. Also, unambiguous evidence for the conversions of the dehydratase product, UDP-α-d-4-keto-4,6-deoxy-GlcNAc, to UDP-α-d-QuiNAc was obtained using real-time 1H-NMR spectroscopy and mass spectrometry. The two genes overlap by 4 nucleotides and similar operon organization and identical gene sequences were also identified in a few other Bacillus species suggesting they may have similar roles in the lifecycle of this class of bacteria important to human health. Our results provide new information about the ability of Bacilli to form UDP-QuiNAc and will provide insight to evaluate their role in the biology of Bacillus.