Insertion of a Loop Structure into the "Loopless" GH19 Chitinase from Bryum coronatum.

Chitinases belonging to the GH19 family have diverse loop structure arrangements. A GH19 chitinase from rye seeds (RSC-c) has a full set of (six) loop structures that form an extended binding cleft from -4 to +4 ("loopful"), while that from moss (BcChi-A) lacks several loops and forms a shortened binding cleft from -2 to +2 ("loopless"). We herein inserted a loop involved in sugar residue binding at subsites +3 and +4 of RSC-c (Loop-II) into BcChi-A (BcChi-A+L-II), and the thermal stability and enzymatic activity of BcChi-A+L-II were then characterized and compared with those of BcChi-A. The transition temperature of thermal unfolding decreased from 77.2 ˚C (BcChi-A) to 63.3 ˚C (BcChi-A+L-II) by insertion of Loop-II. Enzymatic activities toward the chitin tetramer (GlcNAc)₄ and the polymeric substrate glycol chitin were also suppressed by the Loop-II insertion to 12 and 9 %, respectively. The Loop-II inserted into BcChi-A was found to be markedly flexible and disadvantageous for protein stability and enzymatic activity.