Bacterial Cellulose Synthase Catalyzes the Formation of Chitin

ABSTRACT Cellulose synthases (CesA) are widely considered highly substrate‐specific, exclusively utilizing nucleotide‐sugar donors to produce β‐1,4‐glucan. However, several in vivo studies have reported incorporation of N‐acetylglucosamine (GlcNAc) into cellulose under certain metabolic conditions, raising the question of whether CesA can directly utilize UDP‐α‐D‐GlcNAc as a substrate. Here, the recombinant bacterial cellulose synthase complex from Rhodobacter sphaeroides (rBcsA‐B) was employed to address this question biochemically. rBcsA‐B catalyzes uridine diphosphate (UDP) cleavage from UDP‐α‐D‐GlcNAc, although with reduced binding affinity (Km = 1.7 ± 0.5 mM) and catalytic efficiency (Vmax = 0.013 ± 0.008 µM NADH/s) relative to natural substrate UDP‐α‐D‐Glc (Km = 0.7 ± 0.2 mM; Vmax = 0.067 ± 0.02 µM NADH/s). Raman spectroscopic analysis of the reaction pellet revealed bands at 890, 1099, 1372, and 1673 cm − 1 , consistent with glycosidic bonds in β‐chitin conformation. Active site mutagenesis of residues within the QxxRW and FxVTxK motifs, namely R382A, Q379A, and K508A, showed reduced catalytic activity toward both substrates. Whereas R382A and Q379A retained donor‐substrate discrimination, K508A did not, suggesting that K508 contributes specifically to discrimination between the C2‐hydroxyl of UDP‐α‐D‐Glc and the bulkier N‐acetyl group of UDP‐α‐D‐GlcNAc, providing direct biochemical evidence that cellulose synthase possesses an intrinsic capacity to synthesize chitin.