Selection of beta-lactamases and penicillin binding mutants from a libraryof 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 w as generated in the region encompassing residues 163 to 171 of the active s ite Omega-loop. Two in vitro selection protocols were designed to extract f rom the library phage-enzymes characterised by a fast acylation by benzyl-p enicillin (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 streptav idin. As labelling with biotinylated suicide substrates tends to select enz ymes that do not turnover, a counter-selection against penicillin binding m utants was introduced to extract the beta-lactamases. The selected phage-en zymes were characterised by sequencing to identify conserved residues and b y kinetic analysis of the reaction with benzyl-penicillin. Several penicill in binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised b y biphasic kinetics. These data are interpreted by a kinetic scheme in whic h the enzymes exist in two interconvertible conformations. The rate constan t of the conformational change suggests that it involves an isomerisation o f 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. (C) 2000 Academic Press.