Sensitivity of family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the T33I mutant to Oryza sativa xylanase inhibitor protein (OsXIP): An experimental and computational study

Mingqi Liu; Jiayi Li; Ashfaq Ur Rehman; Shenggan Luo; Yuting Wang; Huiwen Wei; Keer Zhang

doi:10.1016/j.enzmictec.2022.109998

Sensitivity of family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the T33I mutant to Oryza sativa xylanase inhibitor protein (OsXIP): An experimental and computational study

Enzyme Microb Technol. 2022 May:156:109998. doi: 10.1016/j.enzmictec.2022.109998. Epub 2022 Jan 29.

Authors

Mingqi Liu¹, Jiayi Li², Ashfaq Ur Rehman³, Shenggan Luo³, Yuting Wang⁴, Huiwen Wei⁴, Keer Zhang⁴

Affiliations

¹ Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou 310018, China. Electronic address: 07a0904048@cjlu.edu.cn.
² State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: jemmylee@sjtu.edu.cn.
³ State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
⁴ Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou 310018, China.

PMID: 35121218
DOI: 10.1016/j.enzmictec.2022.109998

Abstract

Glycoside hydrolase (GH) family 10 and 11 xylanases are inhibited by many xylanase inhibitor proteins (XIPs). We recombinantly expressed the Oryza sativa xylanase inhibitor protein (OsXIP) in Pichia pastoris GS115, with a molecular mass of 47.0 kDa. Family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the mutant T33I (DS199) were inhibited by the recombinant OsXIP (rePOsXIP) through competive inhibition, with corresponding inhibition constants (K_i) of 54.09 and 12.16 nM. After incubation with rePOsXIP (70 nM) at 40 °C for 40 min, inhibitory rates of reBaxA and DS199 (0.2 U) were 23.7% and 76.7%, respectively. Xylooligosaccharides with low concentration were released from beechwood xylan by reBaxA and DS199 in the presence of reOsXIP. Intrinsic fluorescences of reBaxA and DS199 were statically quenched by rePOsXIP in a concentration-dependent manner. Molecular dynamics (MD) simulations and conformational analysis of OsXIP-BaxA and OsXIP-DS199 revealed that the long loop (Lα₄β₅) of OsXIP inserted into the catalytic grooves of BaxA and DS199. The DS199 enhanced the binding affinity to OsXIP, causing conformational alterations on protein-protein interface residues, thereby forming more hydrogen bonds and van der Waals forces. MM/GBSA analysis revealed that the binding free energy (∆G_bind) of OsXIP-DS199 was enhanced by 2.08 kcal/mol compared to that of OsXIP-BaxA. The OsXIP binding induced a conformational changes among residues in the cord and thumb regions of BaxA and DS199. In particular, the T¹¹¹RYNAP¹¹⁶ residues in the thumb region of DS199 was maintained close to OsXIP by specific bonds. Additional MD simulations revealed that Y113A or T93A mutation of BaxA suppressed the binding affinity by diminishing interface associations of OsXIP-BaxA. This study partially elucidats the molecular basis of inhibitory mechanism and structure-function relationships of GH11 xylanases. Our findings inform rational designs of mutant xylanases with higher resistance to inhibitor proteins.

Keywords: Molecular dynamics (MD) simulations; Oryza sativa xylanase inhibitor protein (OsXIP); Protein-protein interactions; Xylanase.

MeSH terms

Bacillus amyloliquefaciens* / genetics
Bacillus amyloliquefaciens* / metabolism
Endo-1,4-beta Xylanases / metabolism
Oryza* / metabolism
Recombinant Proteins / genetics
Xylans / metabolism

Substances

Recombinant Proteins
Xylans
Endo-1,4-beta Xylanases