Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7

Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coli O55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an alpha 1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Gal beta 1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-L-Fuc (k(cat)/K-m = 9.2 min(-1) mM(-1)) as that toward GDP-colitose (k(cat)/K-m = 12 min(-1) mM(-1)). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.