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A heat-labile beta-lactamase has been purified from culture supernatants of Psychrobacter immobilis A5 grown at 4 degrees C and the corresponding chromosomal ampC gene has been cloned and sequenced. All structural and kinetic properties clearly relate this enzyme to class C beta-lactamases. The kinetic parameters of P. immobilis beta-lactamase for the hydrolysis of some beta-lactam antibiotics are in the same range as the values recorded for the highly specialized cephalosporinases from pathogenic mesophilic bacteria. By contrast, the enzyme displays a low apparent optimum temperature of activity and a reduced thermal stability. Structural factors responsible for the latter property were analysed from the three-dimensional structure built by homology modelling. The deletion of proline residues in loops, the low number of arginine-mediated H-bonds and aromatic-aromatic interactions, the lower global hydrophobicity and the improved solvent interactions through additional surface acidic residues appear to be the main determinants of the enzyme flexibility.

The interactions between imipenem and four monobactams and three class A beta-lactamases have been studied in detail. Despite their reputation as being beta-lactamase-stable, some of these compounds were significantly hydrolysed by the enzymes. The results obtained with the Streptomyces albus G beta-lactamase have been analysed in the light of molecular modelling studies. The discussion is extended to include other so-called beta-lactamase-stable antibiotics to demonstrate that this appellation can often be misleading.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.