A PUTATIVE PATHWAY FOR BIOSYNTHESIS OF THE O-ANTIGEN COMPONENT, 3-DEOXY-L-GLYCERO-TETRONIC ACID, BASED ON THE SEQUENCE OF THE VIBRIO-CHOLERAE O1 RFB REGION

The nucleotide sequence of a region of the rfb genes, encoding biosynt hesis of the Vibrio cholerae (Vc) O1 O-antigen, was determined. Analys is of the open reading frames (ORFs) within this region has revealed s imilarities with a number of different classes of biosynthetic protein s and enzymes. The ORFs have been designated RfbK, RfbL, RfbM, RfbN an d RfbO, RfbK is a small, acidic protein which has similarity to the fa mily of proteins known as acyl-carrier proteins (ACP). The RfbL protei n has similarity to a super-family of enzymes which adenylate their su bstrates as a part of their reaction mechanism. Included in these are several acetyl-CoA ligases. Alignment of RfbL with these proteins reve als a highly conserved domain containing the motif GlyXaaXaaGlyXaaPro. This resembles the ATP-binding site motif and may represent a variant of the usual motif, except that Pro replaces Gly. The VcRfbM protein has similarity with a family of long-chain, iron-containing alcohol de hydrogenases, of which the Escherichia coli K-12 fucO and adhE gene pr oducts are also members. The RfbN protein has sequence homology with L uxE and LuxC of Vibrio harveyi (Vh) and other bioluminescent bacterial species. The latter are two components of the enzyme complex which sy nthesizes the long-chain aldehyde used in the V. harveyi bioluminescen ce system. Finally, the VcRfbO protein has sequence similarity with ac etyl-CoA transferases. We were able to identify a number of the gene p roducts using a T7 expression system, confirming several of the alloca ted ORFs. A biosynthetic pathway for the Vc O-antigen component 3-deox y-L-glycero-tetronic acid, based on the enzymatic functions predicted for the RfbK, RfbL, RfbM, RfbN and RfbO proteins, is presented.