title: Binding and processing of β-lactam antibiotics by the transpeptidase LdtMt2from Mycobacterium tuberculosis.
abstract: β-lactam antibiotics represent a novel direction in the chemotherapy of tuberculosis that brings the peptidoglycan layer of the complex mycobacterial cell wall in focus as a therapeutic target. Peptidoglycan stability in Mycobacterium tuberculosis, especially during infection, relies on the nonconventional peptide cross-links formed by l,d-transpeptidases. These enzymes are known to be inhibited by β-lactams, primarily carbapenems, leading to a stable covalent modification at the enzyme active site. A panel of 16 β-lactam antibiotics was characterized by inhibition kinetics, mass spectrometry, and x-ray crystallography to identify efficient compounds and study their action on the essential transpeptidase, LdtMt2. Members of the carbapenem class displayed fast binding kinetics, but faropenem, a penem type compound showed a three to four time higher rate in the adduct formation. In three cases, mass spectrometry indicated that carbapenems may undergo decarboxylation, while faropenem decomposition following the acylation step results in a small 87 Da β-OH-butyryl adduct bound at the catalytic cysteine residue. The crystal structure of LdtMt2at 1.54 Å resolution with this fragment bound revealed that the protein adopts a closed conformation that shields the thioester bond from the solvent, which is in line with the high stability of this dead-end complex observed also in biochemical assays.
  
  
  
  Structural data are available in Protein Data Bank under the accession numbers 5LB1 and 5LBG.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Eva Maria
authors.initials: EM
authors.lastname: Steiner
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Gunter
authors.initials: G
authors.lastname: Schneider
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Robert
authors.initials: R
authors.lastname: Schnell
mesh_terms: D015780:Carbapenems / Q000737:chemistry / Q000627:therapeutic use; D018360:Crystallography, X-Ray; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000472:pathogenicity; D010457:Peptidoglycan / Q000737:chemistry; D010458:Peptidyl Transferases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D014376:Tuberculosis / Q000188:drug therapy* / Q000382:microbiology; D047090:beta-Lactams / Q000737:chemistry* / Q000627:therapeutic use
chemical_list: D015780:Carbapenems; D010457:Peptidoglycan; D047090:beta-Lactams; D010458:Peptidyl Transferases; C107057:fropenem
keywords: covalent adduct; faropenem; l,d-transpeptidase; protein structure; β-lactam antibiotic