The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design

Abstract

Background: Streptococcus pneumoniae is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years. Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against S. pneumoniae and other bacterial pathogens.

Results: Here we report the crystal structure of alanine racemase from S. pneumoniae (AlrSP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3121 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = β = 90° and γ = 120°. Structural comparisons show that AlrSP shares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway.

Conclusions: We have solved the structure of AlrSP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.

Figure 1

Structure of alanine racemase from S. pneumoniae. (A) Ribbon diagram of the alanine racemase monomer with β-sheets colored green and α-helices colored gold. (B) Ribbon diagram of the alanine racemase dimer where one monomer is colored blue and the opposite monomer red. The N'-pyridoxyl-lysine-5'-monophosphate or LLP residue (PLP cofactor covalently bound to lysine; black or grey spheres) resides in the α/β barrel domain of the active site. The active site is composed of residues from the α/β barrel domain of one monomer and residues from the β-strand domain of the other monomer.

Figure 2

Structure-based sequence alignment of the five solved alanine racemase structures from Gram-positive bacteria. Structures are from S. pneumoniae, G. stearothermophilus [29], E. faecalis [38], B. anthracis [36] and S. lavendulae [33]. The black box encloses the conserved PLP binding site, the asterisks (*) mark the PLP-bound Lys residue and the catalytic Tyr residue, the diamond (♦) marks the location of the carbamylated Lys residue, and the residues constituting the entryway to the active site are marked with either I (inner layer) or M (middle layer). Residues that form intermonomer interfaces are highlighted in light green. The purple shading is proportional to the degree of sequence identity across the alignment.

Figure 3

Superposition of alanine racemase monomers from Gram-positive bacteria. (A) Cα atom traces of alanine racemases from G. stearothermophilus (yellow) [29], E. faecalis (green) [38], B. anthracis (blue) [36], S. lavendulae (red) [33], and S. pneumoniae (pink). The superposed N-terminal α/β barrel domains are oriented on the bottom of the picture and the C-terminal β-strand domains on the top. Spheres represent the three structurally equivalent residues used to measure the hinge angle in each structure. The double-headed arrow indicates the variation between hinge angles. The PLP-bound Lys residue from Alr_SP is shown in black. (B) Superposed ribbon representations of the N-terminal domains from E. faecalis (green) [38] and S. pneumoniae (pink), with the most divergent regions colored orange.

Figure 4

Active site of alanine racemase from S. pneumoniae. (A) Electron density 2F_o-F_c map of the active site contoured at 1.5σ, excluding solvent. Residues from the first monomer are colored pink, residues from the second monomer are blue and are denoted with primed numbers. The PLP-bound Lys residue (LLP) is grey. (B) Superposition of the active site residues from Gram-positive alanine racemase structures with Alr_SP; only S. pneumoniae residues are labeled. Residues pictured are from G. stearothermophilus (yellow) [29], E. faecalis (green) [38], B. anthracis (blue) [36], S. lavendulae (red) [33], and S. pneumoniae (pink). The chloride ion from the B. anthracis structure is depicted as a blue sphere. (C) Unmodeled electron density (green) found in the active site. 2F_o-F_c (light blue) and F_o-F_c (green and red) maps are contoured at 1.5 and 3.0 σ, respectively. Residues are colored and labeled as described for Figure 4A.

Figure 5

Schematic diagram of polar interactions around PLP in the active site of alanine racemase from S. pneumoniae. For clarity, interactions with water molecules have not been included. Primed numbers denote residues from the second monomer. This figure was drawn after LeMagueres et al. [32].

Figure 6

Molecular surface representations of the entryway to the active site of alanine racemase from S. pneumoniae. (A) The surface of three layers of entryway residues: residues comprising the inner layer are pink (here, the constricting Tyr352 and Tyr263' residues can be seen), the middle layer residues are orange, and the outer layer residues are blue. The PLP cofactor is colored green. Primed numbers denote residues from the second monomer. (B) Surface of the entryway colored by electrostatic potential (same view as in A).

Figure 7

Pentagonal ring waters near the substrate binding site in alanine racemase from S. pneumoniae. The electron density 2F_o-F_c map is contoured at 0.8σ. Residues are shown as sticks, red spheres represent water molecules, and dashed yellow lines represent hydrogen bonds. Residues from the first monomer are colored pink, residues from the second monomer are blue and are denoted with primed numbers. The PLP-bound Lys residue (LLP) is grey. For simplicity, only some of the residues are shown.