| Summary: | <p>Abstract</p> <p>Background</p> <p>Chitin synthase 3a (CHS3a) from <it>Botrytis cinerea </it>(Bc) catalyses the multiple transfer of <it>N</it>-acetylglucosamine (GlcNAc) residues to the growing chitin chain. Chitin, a β-1,4 linked GlcNAc homopolymer, is an essential cell wall component of filamentous fungi. Chitin synthase, processive membranous protein, has been recognized as a promising target for new antifungicides. Enzymatic characterizations of chitin synthases have been limited, mainly because purity and amounts of integral enzyme obtained after purification procedures have not been sufficient.</p> <p>Findings</p> <p>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 <it>Escherichia coli</it>. 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.</p> <p>Conclusions</p> <p>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.</p>
|
|---|
