| With multi-drug resistant tuberculosis cases increasing globally, the need for better antibiotics and new targets for the development of novel anti-tuberculosis drugs is becoming more urgent. beta-lactams, antibiotics that are known to target bacterial D,D-transpeptidases and impair the bacterial cell wall formation and maintenance, have not been used successfully to treat tuberculosis. These enzymes carry out the ubiquitous (4→3) cross-linking of peptide stems to form the mesh of the peptidoglycan-layer. The peptidoglycan of Mycobacterium tuberculosis is also cross-linked with 3→3 linkages and they play an important role in the cellular physiology of Mycobacterium tuberculosis and its susceptibility to beta-lactams. The membrane bound L,D-transpeptidase, LdtMt2, was recently identified as one of the enzymes that carries out this cross-link in Mycobacterium tuberculosis . This thesis reports the crystal structure of the extra-membrane portion of LdtMt2 (ex-LdtMt2) at 1.7 A resolution as well as kinetic, calorimetric and mutational studies. The protein is formed by two domains: an YkuD catalytic domain and a N-terminal (stalk) domain with an immunoglobulin fold. The structure shows a fragment of the expression host peptidoglycan tightly bound to the catalytic site. This complex provides insight into the binding of substrates to the enzyme. The structural, kinetic and calorimetric studies reported here provided details about the enzymatic mechanism of LdtMt2 that can be used as the basis for the design of inhibitors that target the activity of the enzyme. |
