Abstract
Acinetobacter baumannii is an opportunistic Gram-negative bacterium, which is a common cause of hospital acquired infections. Numerous antibiotic resistant strains exist, emphasising the need for developing new antimicrobials. Alanine racemase is a pyridoxal 5’-phosphate dependent enzyme, responsible for racemisation between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell wall, its inhibition is lethal to prokaryotes, making it an excellent antibiotic drug target.
In this study, A. baumannii alanine racemase (AlrAba) was cloned and expressed in Escherichia coli. A purification protocol was then developed involving ammonium sulphate fractionation and chromatography steps (Hydrophobic interaction, anion exchange and size exclusion). This purification protocol was able to produce 11.5 mg/L of AlrAba with a purity of greater than 95 %. The kinetic parameters of AlrAba were determined using spectrometric coupled enzyme activity assays. The Vmax and Km for the L-alanine to D-alanine reaction were found to be 220.5 U/mg and 1.56 mM, respectively. The Vmax and Km for the D-alanine to L-alanine reaction were found to be 11.3 U/mg and 0.56 mM, respectively.
AlrAba was successfully crystallised and the structure was determined using X-ray crystallography. The structure was initially solved to 1.9 Å resolution via molecular replacement using the monomer of Pseudomonas aeruginosa alanine racemase as the search model. The final structure had an Rfactor of 19.7 % and an Rfree of 23.4 %. The resolution was then extended to 1.65 Å with an Rfactor of 20.6 % and Rfree of 23.6 %.
The tertiary structure AlrAba was established to be a homodimer, in which the two monomers interact in a head to tail manner, resulting in two active sites per enzyme. Each active site is comprised of residues from the N-terminal domain of one monomer and the C-terminal domain of the second monomer. The N-terminal domain corresponds to residues 1 – 230, and consists of an eight-stranded α/β barrel. The C-terminal domain corresponds to residues 231 – 356, and mainly contains β-strands. Comparison of AlrAba with alanine racemases from closely related bacteria demonstrated a conserved overall fold, substrate entryway and active site. The structure of AlrAba will provide a template for future structure-based drug design studies.