Synergistic effects between the additions of a disulphide bridge and an N-terminal hydrophobic sidechain on the binding pocket tilting and enhanced Xyn11A activity

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Author list: Boonyaputthikul H., Muhammad A., Roekring S., Rattanarojpong T., Khunrae P., Sutthibutpong T.

Publisher: Academic Press Inc.

Publication year: 2019

Volume number: 672

ISSN: 0003-9861

eISSN: 0003-9861

URL: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85070636382&doi=10.1016%2fj.abb.2019.108068&partnerID=40&md5=7d49dd42024ef35d1e06241dc78a7eff

Languages: English-Great Britain (EN-GB)

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Abstract

Synergistic effect of distal site-directed mutations and molecular mechanisms on the enhanced thermostability of GH11 xylanase from B. firmus Strain K-1 (xyn11A) was investigated through enzyme activity assays and atomistic molecular dynamics (MD) simulation. From the experiment, single N-terminal leucine substitution at K40L caused a significant drop in enzymatic activity. However, the addition of a disulphide bond at S100C/N147C, along with the K40L mutation enhanced the enzymatic activity at room temperature. Molecular mechanisms on the improvement of enzymatic activity were addressed through atomistic molecular dynamics (MD) simulations of enzyme-substrate complexes. Conformational analysis of the right-hand-shaped GH11 protein structures showed that K40L mutation ‘tilted’ the Palm region away from the Pinky finger at N-terminus and S100C/N147C tilted the Palm region towards the Pinky finger at N-terminus, which destabilized the binding complexes. The extended hydrophobic cluster formed within the K40L/S100C/N147C mutant stabilized the loops associated with the N-terminus and the Thumb region, which facilitated substrate binding and corresponded to the enhanced activity. This proposed mechanism could serve as a scheme for protein engineering to enhance enzymatic activity of GH11 enzymes at low temperatures. © 2019 Elsevier Inc.

Keywords

GH11 xylanase, molecular dynamics, protein engineering, synergism