title: Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.
abstract: CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.
authors.affiliation: Institute for Protein Research, Osaka University, Osaka, Japan.
authors.forename: Takuji
authors.initials: T
authors.lastname: Oyama
authors.forename: George E
authors.initials: GE
authors.lastname: Schmitz
authors.forename: Dylan
authors.initials: D
authors.lastname: Dodd
authors.forename: Yejun
authors.initials: Y
authors.lastname: Han
authors.forename: Alanna
authors.initials: A
authors.lastname: Burnett
authors.forename: Naoko
authors.initials: N
authors.lastname: Nagasawa
authors.forename: Roderick I
authors.initials: RI
authors.lastname: Mackie
authors.forename: Haruki
authors.initials: H
authors.lastname: Nakamura
authors.forename: Kosuke
authors.initials: K
authors.lastname: Morikawa
authors.forename: Isaac
authors.initials: I
authors.lastname: Cann
mesh_terms: D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D046968:Thermoanaerobacter / Q000201:enzymology / Q000235:genetics*
chemical_list: D006026:Glycoside Hydrolases