The increase in antimicrobial resistance (AMR) has led to an increase in demand for the discovery novel antimicrobials with low resistance. Teixobactin is a recently discovered antibiotic with low resistance and has activity against MRSA. However, the teixobactin is unculturable and consists of D-amino acids, making it difficult and expensive to synthesise. Racemases are a group of enzymes that catalyse a reversible reaction, turning L-amino acids to D-amino acids. In this project a mutant broad-spectrum racemase, Y308A was expressed and purified for crystallisation in complex with amino acids, to produce crystals for X-ray crystallography. The reflection data was used to solve the protein structure by molecular replacement, for analysis of the active site interactions with the pyridoxal-5’-phosphate (PLP) ligand and potentially with a “trapped” amino acid.
Through structural analysis it was discovered that the racemase is a homodimer, with each subunit consisting of 2 domains: an N-terminal domain with 8 α/β barrels and a C-terminal domain consisting of β sheets. Each subunit held an active site in the cleft between the 2 domains, where the PLP ligand sits. Both active sites of the dimer have active site residues within both subunits, making it non-functional as a monomer. The PDB sequence was used in a protein-protein BLAST search to identify the closest structural homologues. Clustal Omega alignments were produced to analyse the conservation of active site residues. The putative catalytic residues were determined to be K74 and Y319, which were conserved across the structural homologues of racemase Y308A.
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