Abstract
Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N'-diacetylchitobiose (GlcNAc2) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc2 and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc2 as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.
Highlights
Chitin synthases are stimulated by N-acetylglucosamine (GlcNAc)
To determine the nature of Chs2 reaction products at higher resolution, we focused on chitin oligosaccharides (COs), which are made by S. cerevisiae chitin synthase (CS) at low UDP-GlcNAc concentrations [9, 12, 28]
GlcNAc Strongly Stimulates Formation of GlcNAc2 and COs— Chs2-overexpressing membranes from chs1⌬ chs3⌬ cells were incubated with fixed amounts of UDP-[14C]GlcNAc and increasing amounts of unlabeled UDP-GlcNAc, and reaction mixtures were fractionated into aqueous-soluble, organicsoluble, and chloroform/methanol/water-insoluble material according to an adaptation of the procedure of Bligh and Dyer [22]
Summary
Chitin synthases are stimulated by N-acetylglucosamine (GlcNAc). Results: GlcNAc and 2-acylamido analogues of GlcNAc stimulate formation of chitin oligosaccharides by yeast chitin synthase, and GlcNAc is transferred to the 2-acylamido analogues. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N-diacetylchitobiose (GlcNAc2) was the major reaction product. We show that formation of COs is strongly dependent on the inclusion of free GlcNAc or certain 2-acylamido analogues of GlcNAc in assays and that Chs can transfer a single GlcNAc from UDP-GlcNAc to 2-acylamido analogues of GlcNAc and extend the resulting disaccharide with further GlcNAc residues
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