title: The loop structure of Actinomycete glycoside hydrolase family 5 mannanases governs substrate recognition.
abstract: Endo-β-1,4-mannanases from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) demonstrated different substrate specificities. StMan hydrolyzed galactosylmannooligosaccharide (GGM5; 6(III) ,6(IV) -α-d-galactosyl mannopentaose) to GGM3 and M2, whereas TfMan hydrolyzed GGM5 to GGM4 and M1. To determine the region involved in the substrate specificity, we constructed chimeric enzymes of StMan and TfMan and evaluated their substrate specificities. Moreover, the crystal structure of the catalytic domain of StMan (StMandC) and the complex structure of the inactive mutant StE273AdC with M6 were solved at resolutions of 1.60 and 1.50 Å, respectively. Structural comparisons of StMandC and the catalytic domain of TfMan lead to the identification of a subsite around -1 in StMandC that could accommodate a galactose branch. These findings demonstrate that the two loops (loop7 and loop8) are responsible for substrate recognition in GH5 actinomycete mannanases. In particular, Trp281 in loop7 of StMan, which is located in a narrow and deep cleft, plays an important role in its affinity toward linear substrates. Asp310 in loop8 of StMan specifically bound to the galactosyl unit in the -1 subsite.
authors.affiliation: Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences (RIBS), Okayama, Japan.|Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Kumagai
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Yamashita
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Tagami
authors.affiliation: Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences (RIBS), Okayama, Japan.
authors.forename: Misugi
authors.initials: M
authors.lastname: Uraji
authors.affiliation: Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences (RIBS), Okayama, Japan.
authors.forename: Kun
authors.initials: K
authors.lastname: Wan
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Okuyama
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo, Japan.|Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
authors.affiliation: Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences (RIBS), Okayama, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Hatanaka
mesh_terms: D039903:Actinobacteria / Q000201:enzymology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D030562:Databases, Protein; D006868:Hydrolysis; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase
keywords: Actinomycete; chimeric enzyme; galactosylmannooligosaccharide; glycoside hydrolase family 5; mannanase