Chitin oligosaccharide binding to a family GH19 chitinase from the moss Bryum coronatum

Authors


T. Fukamizo, Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
Fax: +81 742 73 8976
Tel: +81 742 73 8237
E-mail: fukamizo@nara.kindfai.ac.jp
M. Sørlie, Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway
Fax: +47 64 96 59 01
Tel: +47 64 96 59 02
E-mail: morten.sorlie@umb.no

Abstract

Substrate binding of a family GH19 chitinase from a moss species, Bryum coronatum (BcChi-A, 22 kDa), which is smaller than the 26 kDa family GH19 barley chitinase due to the lack of several loop regions (‘loopless’), was investigated by oligosaccharide digestion, thermal unfolding experiments and isothermal titration calorimetry (ITC). Chitin oligosaccharides [β-1,4-linked oligosaccharides of N-acetylglucosamine with a polymerization degree of n, (GlcNAc)n, = 3–6] were hydrolyzed by BcChi-A at rates in the order (GlcNAc)6 > (GlcNAc)5 > (GlcNAc)4 >> (GlcNAc)3. From thermal unfolding experiments using the inactive BcChi-A mutant (BcChi-A-E61A), in which the catalytic residue Glu61 is mutated to Ala, we found that the transition temperature (Tm) was elevated upon addition of (GlcNAc)n (= 2–6) and that the elevation (ΔTm) was almost proportional to the degree of polymerization of (GlcNAc)n. ITC experiments provided the thermodynamic parameters for binding of (GlcNAc)n (= 3–6) to BcChi-A-E61A, and revealed that the binding was driven by favorable enthalpy changes with unfavorable entropy changes. The change in heat capacity (ΔCp°) for (GlcNAc)6 binding was found to be relatively small (−105 ± 8 cal·K−1·mol−1). The binding free energy changes for (GlcNAc)6, (GlcNAc)5, (GlcNAc)4 and (GlcNAc)3 were determined to be −8.5, −7.9, −6.6 and −5.0 kcal·mol−1, respectively. Taken together, the substrate binding cleft of BcChi-A consists of at least six subsites, in contrast to the four-subsites binding cleft of the ‘loopless’ family 19 chitinase from Streptomyces coelicolor.

Database

Chitinase, EC 3.2.1.14

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