Biochemical characterization of an {alpha}1,2-colitosyltransferase from Escherichia coli O55:H7

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 α1,2-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 (Galβ1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km = 9.2 min–1 mM–1) as that toward GDP-colitose (kcat/Km = 12 min–1 mM–1). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.

Source: Glycobiology - March 29, 2016 Category: Biology Authors: Wu, Z., Zhao, G., Li, T., Qu, J., Guan, W., Wang, J., Ma, C., Li, X., Zhao, W., Wang, P. G., Li, L. Tags: Glycan Synthesis Source Type: research