Priming and elongation of chitin chains: Implications for chitin synthase mechanism

Most fungi have multiple chitin synthases (CSs) that may make chitin at different sites on the cell surface, at different times during growth, and in response to cell wall stress. The structure-based model for CS function is for transfer of GlcNAc from UDP-GlcNAc at the cytoplasmic face of the plasma membrane to the non-reducing end of a growing chitin chain, which is concomitantly translocated through a transmembrane channel formed by the synthase. Two aspects of CS mechanism are investigated: how chains might be initiated, and what governs how long they can get. First, it is shown that CSs incorporate free GlcNAc into di-N-acetylchitobiose and into insoluble chitin in a UDP-GlcNAc-dependent manner, and therefore that GlcNAc primes chitin synthesis. Second, average lengths of insoluble chitin chains were measured by determining the molar ratio of priming GlcNAc to chain-extending, UDP-GlcNAc-derived GlcNAc, and showed dependence on UDP-GlcNAc concentration, approaching a maximum at higher concentrations of substrate. These results, together with previous findings that 2-acylamido GlcN analogues prime formation of chitin oligosaccharides and stimulate chitin synthesis are discussed in the context of the structure-based model, and lead to the following proposals. 1) CSs may “self-prime” by hydrolyzing UDP-GlcNAc to yield GlcNAc. 2) A CS’s active site is not continuously occupied by a nascent chitin chain, rather, CSs can release chitin chains, then re-initiate, and therefore synthesize chitin chains in bursts. 3) The length of chitin chains made by a given CS will impact that CS’s contribution to construction of the fungal cell wall.