Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

Abstract

Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627–4630]. PBP1-Xaa–β-lactamase fusions generated periplasmic β-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [∆M1–R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [∆M1–R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.

Comparative cell wall core biosynthesis in the mycolated pathogens,Mycobacterium tuberculosisandCorynebacterium diphtheriae

“…The Nterminal domain of Class B HMM-PBPs are probably involved in interactions with other membrane proteins [120]. Several PBPs have now been identified in mycobacteria [59,[121][122][123][124][125]. Among these are two putative class A HMM-PBP of M. tuberculosis, encoded by Rv0050 (ponA) and Rv3682.…”

Genetics of Peptidoglycan Biosynthesis

“…The mycobacterial PBPs involved with transglycosylation and D,Dtranspeptidation fall into different classes (10,77). The PonA1 and PonA2 proteins are large, class A PBPs that have both transglycosylation and transpeptidation domains (10,78,79). An M. smegmatis ponA1 transposon mutant was isolated in a screen for mutants with an altered dye binding phenotype, and this mutant had reduced growth rate, altered permeability, and increased susceptibility to β-lactam antibiotics (80).…”