title: Chitin oligosaccharide binding to a family GH19 chitinase from the moss Bryum coronatum.
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), 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 (T(m) ) was elevated upon addition of (GlcNAc)(n) (n = 2-6) and that the elevation (ΔT(m)) was almost proportional to the degree of polymerization of (GlcNAc)(n). ITC experiments provided the thermodynamic parameters for binding of (GlcNAc)(n) (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 (ΔC(p)°) 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.
  
  
  METHODS
  Chitinase, EC 3.2.1.14.
authors.affiliation: Department of Advanced Bioscience, Kinki University, Nakamachi, Nara, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fukuda
authors.forename: Noriko
authors.initials: N
authors.lastname: Kawamoto
authors.forename: Toki
authors.initials: T
authors.lastname: Taira
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D044002:Bryophyta / Q000201:enzymology*; D002151:Calorimetry; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000378:metabolism*; D006868:Hydrolysis; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: D009844:Oligosaccharides; D002686:Chitin; D002688:Chitinases