Chemical synthesis of N-acetylglucosamine derivatives and their use as glycosyl acceptors by the Mesorhizobium loti chitin oligosaccharide synthase NodC

Abstract

Rhizobial bacteria synthesize lipo-chitin oligosaccharide signal molecules (Nod factors) that are essential for the formation of symbiotic organs on the roots of host plants, a process known as nodulation. Biosynthesis of the chitin oligosaccharide moiety in Nod factors is carried out by the rhizobial N-acetylglucosaminyltransferase NodC. The initial acceptor or primer used for the synthesis of chitin oligosaccharides in vivo is unknown. To investigate the acceptor specificity of NodC, we have synthesized derivatives of N-acetylglucosamine (GlcNAc) with different aglycones and tested whether they are acceptors for NodC in vitro using a membrane preparation of an Escherichia coli strain expressing the Mesorhizobium loti chitin oligosaccharide synthase NodC. Analysis of reaction products using thin-layer chromatography shows that GlcNAc derivatives containing simple alkyl chains or other hydrophobic groups linked to C-1 are acceptors for NodC. The enzyme appears to be specific for acceptors in which the aglycone is β-linked. GlcNAc derivatives in which the methyl group of the N-acetyl moiety of GlcNAc is replaced by an allyloxy or benzyloxy group are still used as acceptors by NodC. The original methyl group at this position therefore does not appear to be essential for the interaction between NodC and GlcNAc. A NodC-dependent reaction product that is more hydrophobic than GlcNAc was detected in reaction mixtures containing 5% methanol but lacking an exogenously added acceptor. This may be due to the presence of a natural hydrophobic glycosyl acceptor for NodC in the membranes of E. coli, but the structure of this reaction product remains to be investigated.