title: Structural analysis of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5: implications for adaptation to alkaline conditions.
abstract: Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)(8)-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)(8)-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pK(a) calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline adaptation mechanism.
authors: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors: Yueju
authors: Y
authors: Zhao
authors: Yunhua
authors: Y
authors: Zhang
authors: Yang
authors: Y
authors: Cao
authors: Jianxun
authors: J
authors: Qi
authors: Liangwei
authors: L
authors: Mao
authors: Yanfen
authors: Y
authors: Xue
authors: Feng
authors: F
authors: Gao
authors: Hao
authors: H
authors: Peng
authors: Xiaowei
authors: X
authors: Wang
authors: George F
authors: GF
authors: Gao
authors: Yanhe
authors: Y
authors: Ma
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects*; D000468:Alkalies / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001407:Bacillus / Q000187:drug effects* / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000187:drug effects; D006860:Hydrogen Bonding / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007477:Ions; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000468:Alkalies; D000596:Amino Acids; D001426:Bacterial Proteins; D007477:Ions; D050505:Mutant Proteins; D044902:beta-Mannosidase