Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design

Abstract

Glutamate racemase (RacE) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-gamma-d-glutamate capsule of the pathogen Bacillus anthracis. RacE enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. We have cloned, expressed and purified the two glutamate racemase isozymes, RacE1 and RacE2, from the B. anthracis genome. Through a series of steady-state kinetic studies, and based upon the ability of both RacE1 and RacE2 to catalyze the rapid formation of d-glutamate, we have determined that RacE1 and RacE2 are bona fide isozymes. The X-ray structures of B. anthracis RacE1 and RacE2, in complex with d-glutamate, were determined to resolutions of 1.75 A and 2.0 A. Both enzymes are dimers with monomers arranged in a "tail-to-tail" orientation, similar to the B. subtilis RacE structure, but differing substantially from the Aquifex pyrophilus RacE structure. The differences in quaternary structures produce differences in the active sites of racemases among the various species, which has important implications for structure-based, inhibitor design efforts within this class of enzymes. We found a Val to Ala variance at the entrance of the active site between RacE1 and RacE2, which results in the active site entrance being less sterically hindered for RacE1. Using a series of inhibitors, we show that this variance results in differences in the inhibitory activity against the two isozymes and suggest a strategy for structure-based inhibitor design to obtain broad-spectrum inhibitors for glutamate racemases.

Figure 1

Multiple sequence alignments of RacE. The catalytic cysteines are highlighted in purple and all other strictly conserved residues are highlighted in yellow. The black bar connecting D37 and Y43 (B. anthracis RacE2 sequence numbering) denotes a hydrogen bond that is responsible for maintaining the positioning of loop 4, which is important for substrate recognition.

Figure 2

The pH dependence of RacE1 and RacE2 isozyme reactions using L-glutamate as a substrate. Steady-state kinsetic measurments were made using the circular dichroism assay. (a) The k_cat values of the RacE1 catalysed reaction, tested under conditions of saturating L-glutamate at 25°C, are plotted as a function of pH to determine the pH optimum. (b) The k_cat values of the RacE2 reaction are plotted as a function of pH at both saturating (●) and subsaturating (▲) concentrations of L-glutamate. Data were fit to equation 2 to obtain k_cat,max, pKa1 and pKa2 values (see text).

Figure 3

Stereoview of the final electron density (Fo-Fc) omit map in the active site surrounding the substrate D-glutamate. The electron density is contoured at 3 σ (green) and the product D-glutamate was omitted from the Difference-Fourier calculations.

Figure 4

X-ray structures of RacE isozymes from Bacillus anthracis. (a) Topology diagram for the secondary structure elements of RacE1 and RacE2. Domain 1 is composed of residues 1-95 & 208-270 for RacE2, and 1-98 & 211-276 for RacE1. Domain 2 is composed of residues 96-207 for RacE2, and 99-210 for RacE1. (b) The homodimers are shown with the two chains colored green and yellow. The substrate D-glutamate is shown as space fill model (blue) and is located in the active site opposite the dimer interface (labeled). (c) Close-up view of the dimer interface of RacE2. The two monomers are shown in green and yellow. Amino acid R214 (blue), located in the middle of a helix in one monomer, is hydrogen bonded to the amino acids E215 (red), P99 (magenta) and T103 (cyan) in the other monomer. In RacE1, the corresponding residue R214 is replaced with Ile (I217). This alteration disrupts a total of 6 hydrogen bonds that stabilize the dimer interface between the two monomers.

Figure 5

Comparison of the active sites of B. anthracis RacE2 with that of RacE from A. pyrophilus. (a) RacE2 active site with D-glutamate bound (grey). The conformation of residues H187 and G153 in the RacE2 structure is notably different from that of A. pyrophilus RacE. The possible interactions between the catalytic residues and other residues in the immediate vicinity are shown by dotted lines with distances given in Angstroms. (b) Active site of A. pyrophilus RacE with D-glutamine bound in the reverse orientation with its side chain pointing away from the active site pocket.

Figure 6

Interactions of amino acids within the RacE2 catalytic site with D-glutamate. (a) Potential interactions between the active site residues and the α amino group of D-glutamate. (b) Interactions involved in recognition and stabilization of the Cα-carboxyl group. (c) Interactions likely involved in substrate/product recognition and stabilization of the D-glutamate side chain carboxyl group. A highly conserved loop forms the floor of the active site with D37 forming a hydrogen bond with Y43 that is significant and results in orienting this loop in a conformation that favors substrate binding thereby maintaining an important secondary structure conformation that is involved in substrate recognition. The dashed lines in all panels represent distances between atoms in Å.

Figure 7

Compounds 4 (pink), 8 (blue) and 9 (cyan), docked into the active sites of RacE1 (a) and RacE2 (b) using GOLD. A152 in RacE1 causes the opening to the active site to be less sterically hindered than RacE2, which has V149 at the corresponding position.

Figure 8

Proposed active site model for the racemization of glutamate by B. anthracis RacE isozymes. The illustration is based on the four-location model for protein stereospecificity ; . Three locations including the “threonine pocket” (N75,T76,T186, T121 & Wat6), and the α-NH₃⁺ binding site (D11, T186 and Wat 1), and the R-group –(CH₂)₂-COO⁻ binding site (S12, Y43 & Y44), which help to anchor the substrate, are shown and shaded. The “fourth location” or binding site(s) that are responsible for protonation of a possible ylide-intermediate and hence formation of D-glutamate (Cys185-H187-E153), or L-glutamate (Cys74-D11), are shown.

Figure 9

X-ray Structures of RacE2 (a) and RacE1 (b) with D-glutamate bound in the active site depicted with a solid surface showing the active site pockets. RacE2 has Val at position 149 (shown in magenta) while RacE1 has Ala at the corresponding position. These figures demonstrate the effect that the Val and Ala residues have on the opening and accessibility of the active site pocket, which may have important implications in the design of inhibitors for glutamate racemase enzymes. RacE1 has a more open structure, with the active site Cys more visible (shown by the black arrow) as opposed to the more occluded active site of RacE2 (shown by the white arrow).

Scheme 1

Conditions:(i) Di-tert-butyl dicarbonate, DMAP, Et₃N, CH₂Cl₂. (ii) LHDMS, THF, −78 °C, and then ArCH₂Br. (iii) 2.5 LiOH, THF. (iv) CF₃COOH, CH₂Cl₂, rt. (v) LiBH₄, THF.