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

Mycobacterium leprae has two high-molecular-mass multi-modular penicillin-b inding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, G hosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627-4630]. PBP1-Xaa-beta-lactamase fusi ons generated periplasmic p-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncat ion 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 [Delta M1-R314]PBP1 (PBP1 lacking residues Met-1 to Arg- 314) failed to associate with the membrane, suggesting that the region betw een residues Leu-147 and Arg-314 harbours an additional plasma membrane ass ociation site for PBP1. Truncation of the C-terminus up to 42 residues down stream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that ret ained penicillin-binding activity. [Delta MI-R314]PBP1 could be extracted f rom inclusion bodies and refolded under appropriate conditions to give a fo rm capable of binding penicillin with the same efficiency as full-length PB P1. This is, to the best of our knowledge, the first report of a soluble de rivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-b inding (PB) module of PBP1 was fused at its N-terminal end with the non-pen icillin-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.