Cloning overexpression and purification of mur enzymes of brucella abortus for structural resolution by x-ray crystallography

Abstract

Peptidoglycan’s biosynthesis is one of the most studied mechanisms in microbiology. It consists of two stages, a cytoplasmic phase dependent on the Mur protein family and a periplasmic phase controlled by the Penicillin Binding Proteins (PBPs) responsible for the formation of the bacterial cell wall. The PBPs have been the target for the search and design of antibacterials since the discovery of penicillin. However, with the increasing acquisition of resistance mechanisms by bacteria, introductio n of novel classes of antibiotics is necessary. On this line, drugs have been clinically incorporated which alter significantly other elements of the biosynthetic machinery as the case of fosfomycin, an inhibitory antibiotic of MurA. This protein, along with the rest of the Mur family, is responsible for synthetizing the UDP-N-acetyl-D-muramate pentapeptide (or UDP-MurNAcpentapéptide), the precursor of peptidoglycan. As Mur enzymes are highly conserved in bacteria and there has not been found a crossed reaction with the eukaryotic cells, it is expected that inhibitors of Mur proteins may be potentially broad spectrum bactericidal compounds. On this work we focus on Mur proteins of Brucella abortus, the etiological agent of Mediterranean Fever, a world-wide distribution zoonosis. We have amplified the MurF gene from B. abortus strain with PCR, cloned inside an expression vector pET28a, expressed and purified to homogeneity by QIAGEN© Ni-NTA affinity resin. Now, we are running crystallization assays in order to solve its 3D structure by X-ray diffraction. This would help to the rational design of new inhibitors against brucellosis.