Structural and functional characterisation of glutamate racemase isoform 2 from Acinetobacter baumannii

Abstract

Acinetobacter baumannii is an opportunistic pathogen associated with high patient mortality within the Intensive Care Unit. The spread of antimicrobial resistance has resulted in some strains carrying up to 45 resistance genes, and thus infections with these strains are quickly becoming impossible to treat. Glutamate racemase (MurI) has been recognised as a potential target for new antibiotic development for many bacterial species. This enzyme converts L-glutamate to D-glutamate, a crucial building block for the formation of peptidoglycan. The mechanism and essentiality of the enzyme are conserved over many bacterial genera and species. This study examines the enzyme MurI from A. baumannii and whether it would be a worthy target for future drug development. A. baumannii has two isoforms of MurI, both of which have been shown to be actively transcribed. The focus of this work is isoform 2 (MurIAB2), which has 266 residues and a molecular weight of 29 kDa. MurIAB2 expression, as determined by Western blot, revealed that recombinant E. coli Rosetta 2(DE3) grown at 28°C overnight expressed an encouraging amount of soluble protein. Purification then proceeded using immobilised metal ion chromatography, tobacco etch virus (TEV) cleavage and size exclusion chromatography (SEC), resulting in >95% pure MurIAB2. A coupled enzyme assay using L-glutamate dehydrogenase gave preliminary results indicating that MurIAB2 is active. The Vmax was determined to be 6.948 × 10-3 U.mg-1, and the KM determined to be 36.6 mM. Further assays are required to test potential inhibitors and activators for MurIAB2. Crystallisation trials of MurIAB2 were successful. Optimization of these crystals was also successful and yielded crystals that were suitable for data collection. Data collection of MurIAB2 crystals at the Australian synchrotron yielded four complete data sets with resolutions ranging from 2.0 to 1.4 Å and the assignment of an orthorhombic unit cell. Further data processing and refinement is required to produce a fully refined crystal structure of MurIAB2. Together these results provide novel insight into the structure and function of MurIAB2 and suggest a pathway ahead for in continuing research in this area in hopes of antibiotic development.