Allosteric cooperation in ß-lactam binding to a non-classical transpeptidase

L, D-transpeptidase function predominates in atypical 3→3 transpeptide networking of peptidoglycan (PG) layer in Mycobacterium tuberculosis. Prior studies of L, D39 transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or ß-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by ß-lactams. Here we report identification of an allosteric site at a distance of 21 Å from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second ß-lactam molecule and influences binding at the catalytic site. We provide evidence that two ß-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-pocket and one covalently at the catalytic site. This dual ß-lactam binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new drugs against M. tuberculosis.