The r.m.s. difference between the Cα atoms of the two monomers after superposition is 0.38 Å, and the average B-factors of monomers A and B are 38.4 and 46.9 Å2, respectively. As with other alanine racemases, the AlrSP homodimer contains two active sites, each composed of residues from the

α/β barrel of one Abemaciclib in vitro monomer and residues from the β-strand domain of the other. The pyridoxal phosphate selleck compound (PLP) cofactor is connected to Lys40 through an internal aldimine bond and resides inside the α/β barrel domain. 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. As an incidental finding, the AlrSP structure contained additional electron density within the A monomer, at the end of helix 1 in the N-terminal α/β barrel domain. This planar density resembled a carboxylated aromatic ring, therefore a benzoic acid molecule, which fitted and refined well, was modeled into this region, even though the compound was not added to purification or crystallization conditions Selleck GNS-1480 (topology and parameters obtained from the Hetero-compound Information Centre-Uppsala, HIC-UP [46]). It is situated some distance away from both the active site entryway and the dimer interface. Structural and biochemical comparison with closely related alanine racemases As noted in our previous publication [21], AlrSP displays a high level of sequence similarity with other alanine racemases. The structure-based sequence alignment in Figure 2 demonstrates this similarity

with alanine racemases from other Gram-positive bacteria: AlrEF (which has 52% sequence identity with AlrSP), AlrGS (46% identity), AlrBA (38% identity), and AlrSL (36% identity). Regions absolutely conserved across all of these enzymes include GBA3 the characteristic PLP binding site motif near the N-terminus (AVVKANAYGHG), the two catalytic amino acid residues of the active center (Lys40, Tyr263′; throughout this paper, primed numbers denote residues from the second monomer) and the eight residues making up the entryway to the active site (inner layer: Tyr263′, Tyr352, Tyr282′, and Ala169; middle layer: Arg307′, Ile350, Arg288′, and Asp170). 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.