Chitin Translocation Is Functionally Coupled with Synthesis in Chitin Synthase.

Chitin, an extracellular polysaccharide, is synthesized by membrane-embedded chitin synthase (CHS) utilizing intracellular substrates. The mechanism of the translocation of synthesized chitin across the membrane to extracellular locations remains unresolved. We prove that the chitin synthase from (CHS) is a processive glycosyltransferase, which can rapidly produce and tightly bind with the highly polymerized chitin. We further demonstrate that CHS is a bifunctional enzyme, which is necessary and sufficient to translocate the synthesized chitin. CHS was purified and then reconstituted into proteoliposomes (PLs). The nascent chitin is generated and protected from chitinase degradation unless detergent solubilizes the PLs, showing that CHS translocates the newly produced chitin into the lumen of the PLs. We also attempted to resolve the CHS structure of the synthesized chitin-bound state, although it was not successful; the obtained high-resolution structure of the UDP/Mn-bound state could still assist in describing the characterization of the CHS's transmembrane channel. Consistently, we demonstrate that CHS is indispensable and capable of translocating chitin in a process that is tightly coupled to chitin synthesis.