Arg136 is further positioned in AlrSP by a hydrogen bond to Ser309. Sequences of alanine racemases that contain a lysine in position 129 almost always have an accompanying serine or cysteine residue in the equivalent of position 309 [36]. Recently, the AlrBA structure was found to contain an aspargine residue bound to a chloride ion at the equivalent position of Lys129, which appears to play the same role as the carbamylated Lys of positioning the active site arginine [36]. An alignment of alanine racemase sequences by Couñago et al. revealed that the presence of an aspargine residue can occur at the equivalent position

of Lys129 in AlrSP and is likely to be indicative of an internal chloride within the active site in the place of a carbamylated lysine. Notably this change from Lys to Ser appears to always be accompanied by a threonine at the equivalent position CFTRinh-172 concentration of Ser309, even though the threonine does not directly

interact with the chloride ion. The environments on either side of the pyridine ring of PLP are quite different, as reported previously for AlrGS [29, 33]. The side of the PLP that faces the dimer interface is polar in character, with many hydrophilic amino acid residues (including carbamylated Lys129, Arg136, His165 and Arg218), several water molecules and the hydrogen-bond network. The nonpolar side of PLP, in contact with the α/β barrel, contains several hydrophobic residues Isotretinoin (Val38, Leu83, Leu85 and Phe163), no charged residues and no water molecules. learn more As observed in several other alanine racemase structures [[29, 32, 34, 36]], we identified extra density in the active site of AlrSP adjacent to the PLP cofactor (Figure 4C). The position of this density corresponds to that of the acetate modeled in AlrGS. In other structures, this VX-680 mw location has been reported to contain propionate, alanine phosphonate, and a putative substrate molecule in DadXPA [[28–30, 38]]. Water molecules in the same location are found in the AlrMT and AlrSL structures. After unsuccessfully attempting to model a

variety of small molecules into the extra density, including acetate, we left this region of the model empty. Active site entryway The entryway to the active site in AlrSP comprises the α/β barrel domain of one monomer and residues from the C-terminal domain of the other monomer, and is about 13 Å from the active site C4″” atom of PLP. The entryway has a funnel-like shape, with its widest end towards the outside of the enzyme, narrowing as it approaches the PLP. The highly conserved residues comprising the entryway are distributed in layers beginning at the PLP site (Figures 6A and 6B): charged near the entrance, and mainly hydrophobic near the active site [33, 34]. Mutagenesis has shown that these hydrophobic residues have an important role in controlling the substrate specificity of alanine racemase [51].