Contribution of the two conserved tryptophan residues to the catalytic and structural properties of Proteus mirabilis glutathione S-transferase B1-1

Abstract

PmGSTB1-1 (Proteus mirabilis glutathione S-transferase B1-1) has two tryptophan residues at positions 97 and 164 in each monomer. Structural data for this bacterial enzyme indicated that Trp97 is positioned in the helix a4, whereas Trp164 is located at the bottom of the helix a6 in the xenobiotic-binding site. To elucidate the role of the two tryptophan residues they were replaced by site-directed mutagenesis. Trp97 and Trp164 were mutated to either phenylalanine or alanine. A double mutant was also constructed. The effects of the replacement on the activity, structural properties and antibiotic-binding capacity of the enzymes were examined. On the basis of the results obtained, Trp97 does not seem to be involved in the enzyme active site and structural stabilization. In contrast, different results were achieved for Trp164 mutants. Conservative substitution of the Trp164 with phenylalanine enhanced enzyme activity 10-fold, whereas replacement with alanine enhanced enzyme activity 17-fold. Moreover, the catalytic efficiency for both GSH and 1-chloro-2,4-dinitrobenzene substrates improved. In particular, the catalytic efficiency for 1-chloro-2,4-dinitrobenzene improved for both W164F (Trp164-->Phe) and W164A by factors of 7- and 22-fold respectively. These results are supported by molecular graphic analysis. In fact, W164A presented a more extensive substrate-binding pocket that could allow the substrates to be better accommodated. Furthermore, both Trp164 mutants were significantly more thermolabile than wild-type, suggesting that the substitution of this residue affects the overall stability of the enzyme. Taken together, these results indicate that Trp164 is an important residue of PmGSTB1-1 in the catalytic process as well as for protein stability.

Figure 1. Structures and electrostatic potential surfaces of wild-type and W164A

(A) Structure of a PmGSTB1-1 monomer showing the locations of Trp⁹⁷, Trp¹⁶⁴ and GSH bound to Cys¹⁰. The secondary-structure representation was produced using PyMOL [27]. (B) Trace of the superimposed G-site for wild-type crystal structure (green trace) and W164A homology model (blue trace); GSH (red trace). Electrostatic potential surface representation of wild-type crystal structure (C) and the W164A homology model (D). Negative and positive charges are represented in red and blue respectively. The Figures were generated using the program GRASP [30]. Co-ordinates of PmGSTB1-1 are deposited in the Protein Data Bank with accession code 1PMT.

Figure 2. Fluorescence emission spectra of the wild-type and tryptophan mutant enzymes

Wild-type (○), W164A (◆), W164F (□), W97A (△) and W97F (▽). Protein concentration was 3 μM in 10 mM phosphate buffer (pH 7.0) containing 1 mM EDTA. Excitation was at 280 nm.

Figure 3. Far-UV CD spectra for wild-type and tryptophan mutant enzymes

Protein concentration was 17 μM in 10 mM phosphate buffer (pH 7.0). The spectra were recorded at 25 °C. Wild-type (−), W97F (----), W97A (--—-), W164F (-···-) and W164A (—······—).

Figure 4. GdmCl-induced unfolding/refolding (open and filled symbols respectively) transition curves of wild-type and tryptophan mutants, monitored by changes in enzyme activity

(a) Wild-type (○/●), W164A (△/▲) and W164F (□/■). (b) Wild-type (○/●), W97A (△/▲) and W97F (▽/▼).

Figure 5. Effect of temperature on the stability of wild-type and tryptophan mutants

The enzyme activity at 25 °C was taken as 100%. Wild-type (○), W164A (◆), W164F (□), W97A (△) and W97F (▽).

Figure 6. Fluorescence emission spectra of TNS binding with wild-type and tryptophan mutants

Protein concentration was 3 μM in 10 mM phosphate buffer (pH 7.0) containing 1 mM EDTA. Excitation was at 323 nm. The spectra were corrected for the contribution of free unbound TNS. The inset shows the complete emission spectrum of the W164A mutant.