Revealing of a novel xylose-binding site of Geobacillus stearothermophilus xylanase by directed evolution

• Published in: Journal of biochemistry (Tokyo) 2019-02, Vol.165 (2), p.177-184
• Main Authors: Hegazy, Usama M, El-Khonezy, Mohamed I, Shokeer, Abeer, Abdel-Ghany, Somaia S, Bassuny, Roqaya I, Barakat, Amal Z, Salama, Walaa H, Azouz, Rasha A M, Fahmy, Afaf S
• Format: Article
• Language: English
• Subjects: Binding Sites; Crystallography, X-Ray; Directed Molecular Evolution; Endo-1,4-beta Xylanases - chemistry; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - metabolism; Geobacillus stearothermophilus - enzymology; Geobacillus stearothermophilus - genetics; Models, Molecular; Mutation; Structure-Activity Relationship; Xylose - chemistry; Xylose - metabolism
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvy092

Abstract Xylan saccharification is a key step in many important biotechnological applications. Xylose is the main product of xylan degradation and is a major xylanase inhibitor in a bioreactor; however, xylose-binding site of xylanase is not discovered yet. Evolving of xylose-tolerant xylanase variants will reduce the cost of xylanases in industry. Glycoside hydrolase family-10 thermostable Geobacillus stearothermophilus xylanase XT6 is non-competitively inhibited by xylose with inhibition constant ki equals to 12.2 mM. In the absence of X-ray crystallography of xylanase–xylose complex, unbiased random mutagenesis of the whole xylanase gene was done by error-prone polymerase chain reaction constructing a huge library. Screening a part of the library revealed xylose-tolerant mutants having three mutations, M116I, L131P and L133V, clustered in the N-terminus of α-helix 3. The best xylose-tolerant mutant showed higher ki and catalytic capability than that of the parent by 3.5- and 3-fold, respectively. In addition, kcat increased 4.5-fold and KM decreased 2-fold. The molecular docking of xylose into xylanase XT6 structure showed that xylose binds into a small pocket between N-terminus of α-helices 3 and 4 and close to the three mutations. Mobility of α-helices 3 and 4, which controls catalysis rate, is restricted by xylose binding and increased by these mutations.