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. 2007 May 11;189(14):5265–5275. doi: 10.1128/JB.00352-07

FIG. 5.

FIG. 5.

Site-directed mutagenesis reveals active site residues important for catalysis in both RacE1 and RacE2. (A) Three-dimensional homology models. Homology models for RacE1 and RacE2 were constructed using the Chemical Computing Group's MOE 2006.08. The template for both models was the B. subtilis glutamate racemase-d-glutamate structure (Protein Data Bank accession no. 1ZUW), which was aligned with the sequences for B. anthracis RacE1 (BAS0806) and RacE2 (BAS4379) using the Blosum62 substitution matrix. (B) Demonstration of residues important for catalysis. The two putative catalytic cysteine residues in RacE1 (Cys77 and Cys188) and RacE2 (Cys74 and Cys185) were independently changed to alanine by site-directed mutagenesis and assessed for the capacity to racemize glutamate in the l→d direction. The four mutant enzymes (0.31 μM) or two wild-type enzymes (0.31 μM) were independently incubated in the presence of l-glutamate (5 mM). The differential absorption of CD light by glutamate was constantly monitored for 2.25 h utilizing a J-720 CD spectropolarimeter. (C) Chymotrypsin sensitivity patterns. Chymotrypsin protease sensitivity patterns were generated for the wild-type and mutant forms of RacE1 and RacE2. Wild-type RacE1 (0.13 mM), RacE1 C77A (0.13 mM), and RacE1 C188A (0.13 mM) were incubated in a Tris buffer (50 mM Tris-HCl, 100 mM NaCl, 2 mM DTT; pH 8.0) with various concentrations of chymotrypsin (lane A, 0 μg/ml; lane B, 53 μg/ml; lane C, 213 μg/ml; lane D, 640 μg/ml) at 4°C. Wild-type RacE2 (0.13 mM), RacE2 C74A (0.13 mM), and RacE2 C185A (0.13 mM) were incubated in a Tris buffer (50 mM Tris-HCl, 100 mM NaCl, 2 mM DTT; pH 8.0) with various concentrations of chymotrypsin (lane A, 0 μg/ml; lane B, 10 μg/ml; lane C, 40 μg/ml; lane D, 160 μg/ml) at 4°C. After incubation for 1 h, the reactions were stopped by addition of SDS sample buffer, and the samples were electrophoresed on a 16% SDS-polyacrylamide gel and stained with Coomassie brilliant blue G-250. The experiments in panels B and C were performed three separate times. For each independent experiment, we used wild-type or mutant forms of RacE1 or RacE2 from one of three independent enzyme preparations, as well as assay reagents from one of three independent preparations. Representative data from a single experiment are shown. WT, wild type.