Abstract
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-1,4-mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. AxMan113A had higher 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 because of steric hindrance formed by Lys-238 and Trp-239. 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 with the WT. 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-1,4-mannanase-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.
Highlights
-1,4-Mannanase degrades -1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization
Similar to glycoside hydrolase (GH) families 5 and 26, family 113 members belong to clan GH-A, whose members share a typical (/␣)8-barrel protein-fold (TIM barrel) and a retaining GH double-displacement catalytic mechanism (10 –12); on the other hand, GH family 134 -1,4-mannanases display an inverting GH catalytic mechanism [13, 14]
Our studies provide structural insights into the catalytic mechanism of GH family 113 -1,4-mannanases
Summary
NBRC 15112 (AxMan113A) was cloned and expressed in E. coli (Fig. S1). AxMan113A was 36.2 kDa on SDS-PAGE, and the native molecular mass was 38.8 kDa by gel filtration chromatography (Fig. S2), suggesting that the enzyme is a monomer. The amino acid sequence of AxMan113A showed the highest identity (43%) with the -1,4-mannanase from A. acidocaldarius Tc-12-31 (AaManA; GenBankTM accession number ABG77968.1) (Fig. 1). AxMan113A exhibited maximal activity at pH 6.5 in 50 mM McIlvaine buffer (Fig. S3A) and was stable in a pH range of 5.0–9.0, with over 90% activity (Fig. S3B). Optimal temperature of AxMan113A was 45 °C (Fig. S3C), and the enzyme was stable at temperatures below 45 °C, retaining over 90% activity after incubation for 30 min (Fig. S3D)
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