Engineering glycoside hydrolase stability by the introduction of zinc binding

Abstract

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn²⁺ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.

Keywords: X-ray crystallography; glycoside hydrolases; protein engineering; thermal stability.

Figure 1

Crystal structure of J30 as a cartoon representation with (a) and without (b) the protein surface. J30 wt contains an N-terminal Ig-like domain and a C-­terminal catalytic domain, as is common among GH family 9 members. The side chains of the catalytic amino-acid residues Asp147 and Glu523 are depicted in teal. Also highlighted are the side chains corresponding to the residues mediating Zn²⁺ coordination in the triple mutant J30 CCH. (a) Residues Asp147 and Glu523 are positioned at the active site and in the center of the substrate-binding channel. (b) The active site (red asterisk) and Zn²⁺-introduction site (purple asterisk) are separate sites in proximity to one another. Within the catalytic domain, the (α/α)₆-barrel structure (bottom left) and the four-stranded β-sheet (bottom right) are turned into perspective.

Figure 2

The Zn²⁺-introduction site within the crystal structures of J30 wt and J30 CCH. (a) In J30 wt, a hydrogen bond connects the side chains of His115 and Tyr143. Without interfering, the H^α3 of Gly114 and the side chain of Ala98 point towards them. (b) The crystal structure of J30 CCH shows the successful residue mutations and Zn²⁺ incorporation. The metal ion is coordinated by the side chains of Cys98 (2.3 Å distance), Cys114 (2.4 Å), His115 and His143 (both 2.1 Å).

Figure 3

A cellotetraose molecule from the CtCbhA mutant E795Q (PDB entry 1rq5) fitted into the active site of the crystal structure of J30 wt. The individual rings are marked +2 to −2 according to their relative position with respect to the glycosidic bond that would be cleaved by the action of the side chains of Asp147 and Glu523 (labeled in red). Dashes denote hydrogen bonds immediately involved in the nucleophilic attack of the hydrolyzing water molecule (red sphere). The engineered Zn²⁺ site is at the bottom right and is shown with gray labels. The residue-label font sizes reflect the proximity to the viewer.

Figure 4

J30 CCH exhibits an increased thermal tolerance compared with the wild-type enzyme and Zn²⁺-stripped J30 CCH. (a, b) Differential scanning fluorimetry. The standard errors of triplicates are shown. (a) The introduction of Zn²⁺ increased the calculated T _m by 11.2°C. (b) The thermal tolerance of J30 CCH after incubation with EDTA equaled that of the wild-type enzyme, but could be regained by the addition of Zn²⁺. (c) Circular dichroism. The secondary-structure elements of the triple mutant withstand considerably higher temperatures than those of J30 wt.

Figure 5

Enzyme substrate comparison. J30 wt and J30 CCH follow a distinct activity pattern on selected glucoside derivatives. The absorbance of pNP reflects the hydrolysis of its β-glucosides with one (pNPG), two (pNPC) or three (pNPG3) glucose moieties. 100% refers to positive controls using 3.6 M NaOH.

Figure 6

Enzymatic assays. The increase in thermal tolerance is paralleled by a shift in the catalytic profiles of J30 wt and J30 CCH. The pNPC turnover of both enzyme variants is plotted dependent on reaction temperature and pH, showing an increase in the optimal temperature (contour interval of 0.05). Each data-point triplicate is represented by a diamond, the size of which corresponds to its standard error.