Mutations in PilZ domains of newly discovered bacterial β-(1,3;1,4)-mixed linkage glucan synthases modulate polysaccharide production

β-(1,3;1,4)-Mixed linkage glucans (MLGs) are linear homopolysaccharides composed of β-1,4-linked glucose oligomers interconnected by β-1,3 linkages, representing a structurally and functionally distinct class of polysaccharides with diverse biological origins. Our previous work identified and biochemically characterized MLG synthases from the Gram-positive bacteria Romboutsia ilealis and Clostridium ventriculi, establishing their catalytic role in bacterial MLG biosynthesis. In the present study, we sought to expand the repertoire of bacterial MLG synthases by identifying novel glycosyltransferase family 2 (GT2) enzymes across multiple bacterial taxa. Nine GT2 candidates exhibiting sequence homology to R. ilealis GT2 were expressed heterologously in the engineered Saccharomyces cerevisiae LoGSA strain. Two enzymes, Clostridium nigeriense GT2 (CnGT2) and Clostridium cuniculi GT2 (CcGT2), catalyzed the formation of MLG polymers, albeit at markedly reduced levels relative to R. ilealis GT2. Comparative sequence and structural analyses revealed a critical divergence within the PilZ domain: the substitution of asparagine by aspartic acid in the conserved D/NxSxxG motif. This DxSxxG configuration, analogous to that of bacterial cellulose synthases that require c-di-GMP binding for activation, suggests a distinct allosteric regulatory mechanism. Site-directed mutagenesis of this motif modulated MLG biosynthetic output, establishing it as a key determinant governing GT2 catalytic competence and polysaccharide chain elongation.