Effect of acidic amino acids engineered into the active site cleft of Thermopolyspora flexuosa GH11 xylanase

Abstract

Thermopolyspora flexuosa GH11 xylanase (XYN11A) shows optimal activity at pH 6-7 and 75-80 °C. We studied how mutation to aspartic acid (N46D and V48D) in the vicinity of the catalytic acid/base affects the pH activity of highly thermophilic GH11 xylanase. Both mutations shifted the pH activity profile toward acidic pH. In general, the Km values were lower at pH 4-5 than at pH 6, and in line with this, the rate of hydrolysis of xylotetraose was slightly faster at pH 4 than at pH 6. The N46D mutation and also lower pH in XYN11A increased the hydrolysis of xylotriose. The Km value increased remarkably (from 2.5 to 11.6 mg/mL) because of V48D, which indicates the weakening of binding affinity of the substrate to the active site. Xylotetraose functioned well as a substrate for other enzymes, but with lowered reaction rate for V48D. Both N46D and V48D increased the enzyme inactivation by ionic liquid [emim]OAc. In conclusion, the pH activity profile could be shifted to acidic pH due to an effect from two different directions, but the tightly packed GH11 active site can cause steric problems for the mutations.

Activity and stability of hyperthermostable cellulases and xylanases in ionic liquids

“…In addition to these findings, the experiments showing the effect of surface and active site charges and hydrophobic side chains and their engineering (Nordwald and Kaar 2013;Burney et al 2015;Li and Turunen 2015;Nordwald et al 2015;Johnson and Snow 2017;Zhao et al 2018;Zhou et al 2019; Manna and Ghosh 2020) indicate many ways and positions in the protein structures to engineer properties that make the enzymes more tolerant to hydrophilic ILs.…”

“…Introduction of a negatively charged amino acid (aspartic acid) in the active site of the highly thermophilic GH11 xylanase from Thermopolyspora flexuosa, further decreased the activity in [EMIM]OAc compared to the wild-type enzyme. This was probably caused by the attraction of positively charged [EMIM] þ cation to the active site that leads to increased competitive inhibition of substrate binding by [EMIM]OAc (Li and Turunen 2015). While glycoside hydrolases have glutamic or aspartic acid side chains as catalytic residues, the molecular docking results showed that the highest energy cation binding sites in the active site are typically above the catalytic residues (Chawachart et al 2014;Hebal et al 2020).…”

Inhibition of hyperthermostable xylanases by superbase ionic liquids

“…Many endoxylanases from hyperthermophilic microorganisms show tolerance towards ILs [27,39,40,41]. In general, GH10 xylanases tolerate more 1-ethyl-3methylimidazolium acetate ([EMIM]OAc) and other ILs than GH11 xylanases [39,40,41,42]. However, resistance to protein unfolding in thermostable enzymes is not the only factor in IL tolerance [28,39,40].…”

Exploration of a N-terminal disulfide bridge to improve the thermostability of a GH11 xylanase from <i>Aspergillus niger</i>

“…However, there is a risk that the activity of xylanases could decrease, when the mutated sites on thermostability are located close to the active site canyon (Li and Turunen, 2014). In this study, in order to avoid site-directed mutagenesis on thermostability affecting the active site canyon, the active sites of xylanase XynZF-2 were predicted by PROSITE and verified by site-directed mutagenesis (E103D and E194D).…”