Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop.
A combinatorial library of mutants of the phage displayed TEM-1 lactamase was generated in the region encompassing residues 163 to 171 of the active site Omega-loop. Two in vitro selection protocols were designed to extract from the library phage-enzymes characterised by a fast acylation by benzyl-penicillin (PenG) to yield either stable or very unstable acyl-enzymes. The critical step of the selections was the kinetically controlled labelling of the phages by reaction with either a biotinylated penicillin derivative or a biotinylated penicillin sulfone, i.e. a beta-lactamase suicide substrate; the biotinylated phages were recovered by panning on immobilised streptavidin. As labelling with biotinylated suicide substrates tends to select enzymes that do not turnover, a counter-selection against penicillin binding mutants was introduced to extract the beta-lactamases. The selected phage-enzymes were characterised by sequencing to identify conserved residues and by kinetic analysis of the reaction with benzyl-penicillin. Several penicillin binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised by biphasic kinetics. These data are interpreted by a kinetic scheme in which the enzymes exist in two interconvertible conformations. The rate constant of the conformational change suggests that it involves an isomerisation of the peptide bond between residues 166 and 167 and controls a conformation of the Omega-loop compatible with fast acylation of the active site serine residue.
Vanwetswinkel
S
S
Université Catholique de Louvain, Laboratoire de Biochimie Physique et des Biopolymères, Place L. Pasteur, 1 Bte 1B, Louvain-la-Neuve, B1348, Belgium.
Avalle
B
B
Fastrez
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D006602:Hexosyltransferases; D007700:Kinetics; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D019151:Peptide Library; D010458:Peptidyl Transferases; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D046915:Penicillin-Binding Proteins; D019151:Peptide Library; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1