Isolation, purification and characterization of L,D-transpeptidase 2 from Mycobacterium tuberculosis

Abstract

L,D-transpeptidase 2 from Mycobacterium tuberculosis plays a key role in the formation of nonclassical 3-3 peptidoglycan cross-links in a pathogen's cell wall making it resistant to a broad range of penicillin antibiotics. The conditions of cultivation, isolation, and purification of recombinant L,D-transpeptidase 2 from M. tuberculosis have been optimized in this study. Oxidation of the free SH groups of catalytic cysteine Cys354 is an important factor causing the inactivation of the enzyme, which occurs during both the expression and storage of enzyme preparations. The biochemical characteristics of purified L,D-transpeptidase 2 and L,D-transpeptidase 2 lacking domain A were determined; the kinetic constants of enzyme-catalyzed nitrocefin transformation were evaluated.