Identification of a Key Loop for Tuning Transglycosylation Activity in the Substrate-Binding Region of a Chitosanase.

Abstract

Csn-PD, a glycoside family 46 chitosanase from Paenibacillus dendritiformis, exhibits endotype hydrolysis of chitosan and produces (GlcN)2 as the major product. Here, we report the crystal structure of Csn-PD at 1.68 Å resolution. The structure contains 14 α-helices and two β-strands that fold into two globular domains with the substrate bound between them. To evaluate the function of a loop in the substrate-binding region (residues 112-116, NDKHP), a mutant Csn-PDL1, in which this loop was deleted, was generated. Hydrolysis of chitosan by the mutant yielded chitooligosaccharides (COSs) with higher degrees of polymerization (DP) than the wild-type enzyme. Excitingly, (GlcN)6 was produced from smaller COSs via transglycosylation activity of the mutant. Hence, the catalytic performance of a chitosanase was altered by modification of a loop in the substrate-binding regions. Our novel data on a chitosanase with transglycosylation activity offer a promising way to produce COSs with high DP.