Abstract
The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (k cat/K m) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.
Highlights
A disulfide bridge is formed by the oxidation of two thiols each from two cysteines, linking the two cysteines and their respective main peptide chains, which can restrict the motion of the unfolded, random coil of protein or stabilize the folded state of protein [1,2]
Because the rigidity of a protein was positively related to its thermostability [38], the variant AuFaeAA126C-N152C was predicted to be more thermostable than the wild-type AufaeA encoding a mesophilic type A FAE (AuFaeA)
Effects of disulfide bridges on the thermostability of AuFaeA were investigated by either introducing an extra disulfide bridge to, or by eliminating each native disulfide bridge from AuFaeA
Summary
A disulfide bridge is formed by the oxidation of two thiols each from two cysteines, linking the two cysteines and their respective main peptide chains, which can restrict the motion of the unfolded, random coil of protein or stabilize the folded state of protein [1,2]. To predict the thermostability of AuFaeA and its candidate variants with extra disulfide bridges, their 3D structures were modeled and subjected to molecular dynamics (MD) simulation processes, respectively, at 500 K for 10 ns using GROMACS 4.5 package (http:// www.gromacs.org/). Based on the computational prediction, AuFaeAA126C-N152C, a variant of AuFaeA, with the smallest RMSD value was selected. The RMSD value is an important index for evaluating the conformational flexibility of protein at high temperature [29]. The distributions of RMSD values of AuFaeAA126C-N152C, AuFaeAY122C-Y125C and AuFaeA were statistically analyzed and were doi:10.1371/journal.pone.0126864.g004 mainly concentrated on 0.475 Å, 0.825 Å and 0.525 Å, respectively (Fig 3B). Because the rigidity of a protein was positively related to its thermostability [38], the variant AuFaeAA126C-N152C was predicted to be more thermostable than the wild-type AuFaeA. After site-directed mutagenesis, the mutant gene was obtained and confirmed by DNA sequencing
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