O-repeating unit biosynthesis is the first committed step in lipopolysaccharide (LPS) biosynthesis in a variety of gram-negative bacteria. The wbnH gene was previously proposed to encode a glycosyltransferase involved in O-repeating unit synthesis in Escherichia coli O86:H2 strain. In this work, we provide biochemical evidence to show that wbnH encodes a N-acetylgalactosaminyl transferase (GalNAcT) that catalyzes the transfer of GalNAc from UDP-GalNAc to the GalNAc-pyrophosphate-lipid acceptor. WbnH activity was characterized using a synthetic acceptor Substrate GalNAc alpha-PP-O(CH2)(11)-OPh. The resulting disaccharide product GalNAc-alpha-1,3-GalNAc alpha-PP-O(CH2)(11)-OPh was analyzed by LC-MS and NMR spectroscopy. Substrate specificity Study indicates that pyrophosphate and hydrophobic lipid moiety are structural requirements for WbnH activity. Divalent metal cations are not required for enzyme catalysis, suggesting WbnH belongs to glycosyltransferase GT-B3 superfamily. Our results complete the characterization of O86 O-unit assembly pathway, and provide the access of chemically defined O-unit substrates for the further investigation of O-antigen biosynthetic mechanism. (c) 2006 Elsevier Inc. All rights reserved.