Structural and biochemical insights into the substrate-binding mechanism of a novel glycoside hydrolase family 134 β-mannanase

Publication date: Available online 14 March 2018 Source:Biochimica et Biophysica Acta (BBA) - General Subjects Author(s): Xin You, Zhen Qin, Yan-Xiao Li, Qiao-Juan Yan, Bin Li, Zheng-Qiang Jiang Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. β-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-β-mannanase from Rhizopus microsporus (RmMan134A) was cloned and expressed. The recombinant RmMan134A showed maximal activity at pH 5.0 and 50 °C, and exhibited high specific activity towards locust bean gum (2337 U/mg). To gain insight into the substrate-binding mechanism of RmMan134A, four complex structures (RmMan134A–M3, RmMan134A-M4, RmMan134A-M5 and RmMan134A-M6) were further solved. These structures showed that there were at least seven subsites (−3 to +4) in the catalytic groove of RmMan134A. Mannose in the −1 subsite hydrogen bonded with His113 and Tyr131, revealing a unique conformation. Lys48 and Val159 formed steric hindrance, which impeded to bond with galactose branches. In addition, the various binding modes of RmMan134A–M5 indicated that subsites −2 to +2 are indispensable during the hydrolytic process. The structure of RmMan134A–M4 showed that mannotetrose only binds at subsites +1 to +4, and RmMan134A could therefore not hydrolyze mannan oligosaccharides with degree of polymerization ≤4. Through rational design, the specific activity and opt...

Source: Biochimica et Biophysica Acta (BBA) General Subjects - March 15, 2018 Category: Biochemistry Source Type: research