Tuberculosis has attracted increased attention worldwide due to its high morality and its resistance to treatment with traditional antibacterial drugs. The L,D-transpeptidase Ldt(Mt2) confers resistance to traditional beta-lactams and is considered a target for anti-Tuberculosis treatment. Carbapenems are proposed to inhibit Mycobacterium tuberculosis by repressing the activity of Ldt(Mt2). The interaction mechanisms between Ldt(Mt2) and carbapenems have been revealed by Ldt(Mt2) -carbapenem adduct structures along with various biochemical assays. Interestingly, the lack of the 1-beta-methyl group in imipenem may be related to its high binding ability to Ldt(Mt2) . However, there is limited evidence on the interaction mode of Ldt(Mt2) and panipenem, another carbapenem lacking the 1-beta-methyl group. Herein, we identified the biochemical features of panipenem binding to Ldt(Mt2) . We further suggest that the presence of the 1-beta-methyl group in carbapenems is indeed related to the ligand affinity of Ldt(Mt2) and that the presence of the Y308 and Y318 residues in Ldt(Mt2) stabilized the conformation of the Ldt(Mt2) -carbepenem adduct. Our research provides a structural basis for the development of novel carbapenems against L,D-transpeptidases. (C) 2019 Elsevier Inc. All rights reserved.