Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae.
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-beta-lactamase fusions generated periplasmic beta-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 [DeltaM1-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. [DeltaM1-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.
Mahapatra
S
S
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, 700 009 Calcutta, India.
Bhakta
S
S
Ahamed
J
J
Basu
J
J
D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006602:Hexosyltransferases; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000378:metabolism*; D009166:Mycobacterium leprae / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D012995:Solubility; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase