Functional comparison of the two Bacillus anthracis glutamate racemases - PubMed (original) (raw)

Functional comparison of the two Bacillus anthracis glutamate racemases

Dylan Dodd et al. J Bacteriol. 2007 Jul.

Abstract

Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because L-glutamate stereoisomerization to D-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of L-glutamate and D-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of L-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features.

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Figures

FIG. 1.

B. anthracis RacE1 and RacE2 possess significant sequence homology to B. subtilis RacE. Primary amino acid sequences for B. anthracis RacE1 and RacE2 and B. subtilis RacE were aligned utilizing ESPript V.2.2 (

http://espript.ibcp.fr/ESPript/ESPript/

). Secondary structural elements and solvent accessibility indices were imported from the crystal structure of B. subtilis 168 RacE (Protein Data Bank accession no. 1ZUW) (43). For solvent accessibility indices, the darker shaded regions indicate sections in the folded proteins that are exposed to solvent.

FIG. 2.

RacE1 and RacE2 are both functional enzymes in a cell-free system. (A) Purification of recombinant RacE1 and RacE2. The RacE1 and RacE2 clarified lysates and fractions from nickel-chelate affinity chromatography were analyzed by 12% SDS-PAGE, followed by Coomassie brilliant blue G-250 staining. (B) RacE1 and RacE2 secondary structure. CD spectra in the far-UV region (190 to 260 nm) were recorded for RacE1 and RacE2 (2.7 and 2.4 μM, respectively, both in 50 mM potassium borate buffer; pH 8.0). (C) Gel filtration chromatography. The sizes of purified RacE1 and RacE2 were determined by size exclusion FPLC. The molecular weights (MW) of RacE1 and RacE2 were calculated from the retention times of the peak absorbance by comparison with calibration standards having known molecular weights. (D) Racemization of glutamate in the

→

direction. RacE1 and RacE2 were assessed for the capacity to convert

-glutamate to the corresponding

enantiomer by using CD to directly observe the loss of

-glutamate as it was converted to

-glutamate.

-Glutamate (5 mM) was incubated in the absence or presence of RacE1 or RacE2 (1.3, 0.33, or 0.08 μM), and the CD signal was recorded for 2.25 h. (E) Racemization of glutamate in the

→

direction. RacE1 and RacE2 were assessed for the capacity to catalyze the forward (

-glutamate →

-glutamate) and reverse (

-glutamate →

-glutamate) reactions by CD spectroscopy.

-Glutamate (5 mM) or

-glutamate (5 mM) was incubated in the presence of RacE1 (0.31 μM) or RacE2 (0.31 μM), and the CD signal was recorded for 2.25 h. For panels A to E, at least three separate experiments were performed. For each independent experiment, we used RacE1 or RacE2 from one of three independent enzyme preparations, as well as assay reagents from one of three independent preparations. For panels A to E, representative data from a single experiment are shown. In panel C, the molecular weights are reported as the means ± standard deviations from three independent experiments. mAU, milliabsorbance units.

FIG. 3.

Steady-state kinetic analysis of RacE1 and RacE2. RacE1 (0.78 μM) or RacE2 (0.78 μM) was incubated in a potassium borate buffer (50 mM boric acid, 100 mM KCl, 0.2 mM DTT; pH 8.0) in the presence of various concentrations of

-glutamate (0.1 to 10 mM) (A) or

-glutamate (5 to 200 mM) (B), and the change in magnitude of the CD signal was monitored. (A) Steady-state kinetic parameters for the racemization of glutamate in the

→

direction by RacE1 and RacE2. The data are expressed as initial rate of racemization for RacE1 or RacE2 as a function of the

-glutamate concentration. Steady-state kinetic parameters for RacE1 and RacE2 in the presence of

-glutamate were obtained by applying a nonlinear curve fit to the data. (B) Steady-state kinetic parameters for the racemization of glutamate in the

→

direction by RacE1 and RacE2. The data are expressed as the initial rate of racemization for RacE1 and RacE2 as a function of the

-glutamate concentration. Steady-state kinetic parameters for RacE1 or RacE2 in the presence of

-glutamate were obtained by applying a nonlinear least-squares regression utilizing GraphPad Prisim V4.03. For all studies, at least three independent experiments were performed. For each independent experiment, we used RacE1 or RacE2 from one of three independent enzyme preparations, as well as assay reagents from one of three independent preparations. The symbols indicate the means of the data from three independent experiments, and the error bars indicate the standard deviations of the means.

FIG. 4.

pH dependence of RacE1 and RacE2 catalysis. RacE1 and RacE2 were assayed in buffers having pH values ranging from 6.5 to 9.5 with increments of 0.5 pH unit. Each buffer was prepared with a concentration of glutamate (200 mM) that was fully saturating for RacE1 and nearly saturating (83% saturating) for RacE2, so that the initial rate data would report true _k_cat values. Initial rate data were then obtained for RacE1 (0.78 μM) and RacE2 (0.78 μM) in each of the different buffer formulations by measuring the change in magnitude of the CD signal over time. The data are expressed as the turnover number (_k_cat) as a function of the reaction pH. The symbols indicate the means of the data from three independent experiments. For each independent experiment, we used RacE1 or RacE2 from one of three independent enzyme preparations, as well as assay reagents from one of three independent preparations. The error bars indicate standard deviations of the means.

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-

-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

→

direction. The four mutant enzymes (0.31 μM) or two wild-type enzymes (0.31 μM) were independently incubated in the presence of

-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.

FIG. 6.

Three-dimensional homology models reveal differences in RacE1 and RacE2 fine active site features. Three-dimensional 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 RacE-

-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. (A) RacE1 and RacE2 exhibit differences in the spatial arrangement of active site residues. Residues predicted to be important for catalysis in B. anthracis RacE1 (green) and RacE2 (purple) and B. subtilis RacE (gray) were aligned. The RMSD of the superposition of Cα atoms between B. anthracis RacE1 and B. subtilis RacE is 7.5 Å, while the RMSD between B. anthracis RacE2 and B. subtilis RacE is 2.6 Å. (B and C) Differences in inhibitor docking to the active sites of RacE1 and RacE2. The glutamate racemase inhibitor compound 69 (12) is shown docked within the active site of B. anthracis RacE1 (B) and overlaid with the active site of B. anthracis RacE2 (C). Residues that are present at the entrance to the hydrophobic binding pocket of B. anthracis RacE1 (green) and RacE2 (purple) are shown. Compound 69 was unable to dock within the active site of RacE2, presumably due to the larger side chain of RacE2 V149, which aligns with A152 in RacE1.

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Functional comparison of the two Bacillus anthracis glutamate racemases - PubMed (original) (raw)