Active site dynamics and catalytic mechanism in arabinan hydrolysis catalyzed by GH43 endo-arabinanase from QM/MM molecular dynamics simulation and potential energy surface

Abstract

The endo-1,5-α-L-arabinanases, belonging to glycoside hydrolase family 43 (GH43), catalyse the hydrolysis of α-1,5-arabinofuranosidic bonds in arabinose-containing polysaccharides. These enzymes are proposed targets for industrial and medical applications. Here, molecular dynamics (MD), potential energy surface and free energy (potential of mean force) simulations are undertaken using hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to understand the active site dynamics, catalytic mechanism and the electrostatic influence of active site residues of the GH43 endo-arabinanase from G. stearothermophilus. The calculated results give support to the single-displacement mechanism proposed for the inverting GH43 enzymes: first a proton is transferred from the general acid E201 to the substrate, followed by a nucleophilic attack by water, activated by the general base D27, on the anomer carbon. A conformational change (²E ↔E₃ ↔ ⁴E) in the −1 sugar ring is observed involving a transition state featuring an oxocarbenium ion character. Residues D87, K106, H271 are highlighted as potential targets for future mutation experiments in order to increase the efficiency of the reaction. To our knowledge, this is the first QM/MM study providing molecular insights into the glycosidic bond hydrolysis of a furanoside substrate by an inverting GH in solution.

Communicated by Ramaswamy H. Sarma

Keywords:

• Arabinan
• endo-15-α-L-arabinanases
• enzyme mechanism
• conformational itinerary
• QM/MM
• SCC-DFTB

Acknowledgements

This work has been supported by the Thailand Research Fund (TRF) and University of Phayao through the TRF Mid-Career Research Scholar (Grant No. RSA6280104). WM and JJ gratefully acknowledge the Science Classroom in University Affiliated School (SCiUS) Project (http://scius.most.go.th/) for the infrastructure facilities provided to the Demonstration School University of Phayao. JJ thanks Patchreenart Saparpakorn for her computational time. JO thanks the financial support of Project no. 2018-1.2.1-NKP-2018-00005 which has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the 2018-1.2.1-NKP funding scheme. Financial support by the University of Phayao under the Basic Research Fund (Grant No. FF64-RIB002) is also acknowledged.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work has been supported by TRF (Thailand Research Fund) and University of Phayao – Grant Nos. RSA6280104 and FF64-RIB002.