Abstract
BackgroundChitin synthase 3a (CHS3a) from Botrytis cinerea (Bc) catalyses the multiple transfer of N-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.FindingsWe 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.ConclusionsMilligram-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.
Highlights
Chitin synthase 3a (CHS3a) from Botrytis cinerea (Bc) catalyses the multiple transfer of Nacetylglucosamine (GlcNAc) residues to the growing chitin chain
Milligram-scale quantities of CHS3a-SGC protein with native-like properties such as specific substrate UDP-GlcNAc binding could be obtained. These results are encouraging for subsequent heterologous expression of full-length CHS3a
By taking these results into account, it is conceivable that a small part of chitin synthases (CHS), corresponding to the SGC domain previously described, could be sufficient for catalytic activity, while other domains of the enzyme are implicated in other functions, such as membrane localization, binding to chitin and export of chitin fibers
Summary
A linear b-(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]. CHS2 of Saccharomyces cerevisiae characterization revealed that i) the N-terminally truncated CHS2 of S. cerevisiae exhibits the same enzymatic activity as full size enzyme and ii) the 35 kDa fragment corresponding to the region just before the first transmembrane domain should contain the active site of the enzyme [22] By taking these results into account, it is conceivable that a small part of CHS, corresponding to the SGC domain previously described, could be sufficient for catalytic activity, while other domains of the enzyme are implicated in other functions, such as membrane localization, binding to chitin and export of chitin fibers. To investigate the enzymatic properties of chitin synthase, cloning and expression in E. coli of the BcCHS3a recombinant protein including only SGC domain, devoid of both the non-conserved N-terminus region and the highly hydrophobic transmembrane Cterminus region, called CHS3a-SGC-423, was undertaken. The purification, folding, enzymatic activity and UDP-GlcNAc binding of both CHS3a fragments were investigated
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