title: Enhanced xyloglucan-specific endo-β-1,4-glucanase efficiency in an engineered CBM44-XegA chimera.
abstract: Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.
authors.affiliation: Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Gilvan P
authors.initials: GP
authors.lastname: Furtado
authors.forename: Camila R
authors.initials: CR
authors.lastname: Santos
authors.forename: Rosa L
authors.initials: RL
authors.lastname: Cordeiro
authors.forename: Lucas F
authors.initials: LF
authors.lastname: Ribeiro
authors.forename: Luiz A B
authors.initials: LA
authors.lastname: de Moraes
authors.forename: André R L
authors.initials: AR
authors.lastname: Damásio
authors.forename: Maria de Lourdes T M
authors.initials: Mde L
authors.lastname: Polizeli
authors.forename: Marcos R
authors.initials: MR
authors.lastname: Lourenzoni
authors.forename: Mário T
authors.initials: MT
authors.lastname: Murakami
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005936:Glucans / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D013379:Substrate Specificity; D014961:X-Ray Diffraction; D014990:Xylans / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D005936:Glucans; D014990:Xylans; C029353:xyloglucan; D044644:Endo-1,3(4)-beta-Glucanase