Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase

We undertook the preparation of two BcCHS3a fragment proteins, containing only the central domain and devoid of the N-terminal and transmembrane C-terminal regions. The central domain of CHS3a, named SGC (Spsa GntI Core), is conserved in all UDP-glycosyltransferases and it is believed to contain the active site of the enzyme. CHS3a-SGC protein was totally expressed as inclusion bodies in Escherichia coli. We performed recombinant CHS3a-SGC purification in denaturing conditions, followed by a refolding step. Although circular dichroism spectra clearly exhibited secondary structures of renatured CHS3a-SGC, no chitin synthase activity was detected. Nevertheless CHS3a-SGC proteins show specific binding for the substrate UDP-GlcNAc with a dissociation constant similar to the Michaelis constant and a major contribution of the uracil moiety for recognition was confirmed.Milligram-scale quantities of CHS3a-SGC protein with native-like properties such as specific substrate UDP-GlcNAc binding could be easily obtained. These results are encouraging for subsequent heterologous expression of full-length CHS3a.Chitin, a linear β-(1-4)-linked polymer of N-acetylglucosamine (GlcNAc), is an important structural component of the cell walls of many fungi, with different contents of chitin between species [1]. Chitin is absent from plant and mammalian species. Thus, its biosynthesis is recognized as a valuable and attractive target for the design of fungicides [2]. Chitin is synthesized by chitin synthases (CHS), a multiple membrane isoenzyme family. Analysis of several fungal genomes allowed the classification of yeast CHS, Saccharomyces cerevisiae and Candida albicans, into three classes and those of many filamentous fungi into seven or more classes [3,4]. Results were based on the identification of a conserved domain present in all CHS described. For Botrytis cinerea (Bc), a necrotrophic filamentous fungus which causes grey mould on a wide range of plants, eight distinct CHS genes w