Synergistic effects between the additions of a disulphide bridge and an N-terminal hydrophobic sidechain on the binding pocket tilting and enhanced Xyn11A activity
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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DOI：
10.1016/j.abb.2019.108068
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发表时间：
2019-09-15
影响因子：
3.9
通讯作者：
Sutthibutpong, Thana
中科院分区：
文献类型：
--
作者：
Boonyaputthikul, Harit;Muhammad, Auwal;Sutthibutpong, Thana
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.
