Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase fromAmphibacillus xylanus

Xin You, Zhen Qin, Qiaojuan Yan, Shaoqing Yang, Yanxiao Li, Zhengqiang Jiang
2018 Journal of Biological Chemistry  
β-1,4-Mannanase degrades β-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) family 113 β-1,4-mannanase, from Alicyclobacillus acidocaldarius (AaManA) has been structurally characterized, and no complex structure of enzyme-manno-oligosaccharides from this family has been reported. Here, crystal structures of a GH family 113 β-1,4mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose,
more » ... mannobiose, mannotriose, mannopentaose and mannahexaose were solved. AxMan113A had a stronger affinity for -1 and +1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites (-4 to +2) exist in the groove, but mannose units preferentially occupied subsites -4 to -1 owing to steric hindrance formed by Lys238 and Trp239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity of AxMan113A mutants V139C, N237W, K238A and W239Y was improved by 93.7%, 63.4%, 112.9% and 36.4%, respectively, compared to the wild type. In addition, previously unreported surface binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH family 113 β-1,4mannanase-manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism of GH family 113 members, and provide novel insight into protein engineering of GHs to improve their hydrolysis activity. Mannans are major components of hemicellulose, which is widely distributed in http://www.jbc.org/cgi/
doi:10.1074/jbc.ra118.002363 pmid:29871927 fatcat:z5qhrxrlsfbblbg42a3kvajdwq