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Bacillus thuringiensis is a soil-dwelling Gram positive bacterium that has been utilized as a biopesticide for well over 60 years. It is known to contain flagella that are important for motility. One of the proteins found in flagella is flagellin, which is post-translationally modified by O-glycosylation with derivatives of pseudaminic acid. The biosynthetic pathway for the production of CMP-pseudaminic acid in B. thuringiensis, starting with UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), requires seven enzymes. Here, we report the three-dimensional structures of Pen and Pal, which catalyze the first and second steps, respectively. Pen contains a tightly bound NADP(H) cofactor whereas Pal is isolated with bound NAD(H). For the X-ray analysis of Pen, the site-directed D128N/K129A mutant variant was prepared in order to trap its substrate, UDP-GlcNAc, into the active site. Pen adopts a hexameric quaternary structure with each subunit showing the bilobal architecture observed for members of the short-chain dehydrogenase/reductase superfamily. The hexameric quaternary structure is atypical for most members of the superfamily. The structure of Pal was determined in the presence of UDP. Pal adopts the more typical dimeric quaternary structure. Taken together, Pen and Pal catalyze the conversion of UDP-GlcNAc to UDP-4-keto-6-deoxy-l-N-acetylaltrosamine. Strikingly, in Gram negative bacteria such as Campylobacter jejuni and Helicobacter pylori, only a single enzyme (FlaA1) is required for the production of UDP-4-keto-6-deoxy-l-N-acetylaltrosamine. A comparison of Pen and Pal with FlaA1 reveals differences that may explain why FlaA1 is a bifunctional enzyme whereas Pen and Pal catalyze the individual steps leading to the formation of the UDP-sugar product. This investigation represents the first structural analysis of the enzymes in B. thuringiensis that are required for CMP-pseudaminic acid formation.

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Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa-bridged enediyne with two attached sugars, L-mycarose, and L-kedarosamine. The biosynthesis of L-kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N-methyltransferase that utilizes S-adenosylmethionine as the methyl donor and a dTDP-linked C-4 0 amino sugar as the substrate. Here we describe the threedimensional architecture of KedS8 in complex with S-adenosylhomocysteine. The structure was solved to 2.0 Å resolution and refined to an overall R-factor of 17.1%. Unlike that observed for other sugar N-methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of b-strands at the N-termini of its subunits. The structure presented here represents the first example of an N-methyltransferase that functions on C-4 0 rather than C-3 0 amino sugars.

No abstract