On functional and structural heterogeneity of VIM-type metallo-beta-lactamases.
The VIM metallo-beta-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens. In this work a T7-based expression system for overproduction of the VIM-2 enzyme by Escherichia coli was developed, which yielded approximately 80 mg of protein per litre of culture. The enzyme was mostly released into the medium, from which it was recovered at >99% purity by an initial ammonium sulphate precipitation followed by two chromatography steps, with almost 80% efficiency. Determination of kinetic parameters of VIM-2 under the same experimental conditions previously used for VIM-1 (the first VIM-type enzyme detected in clinical isolates, which is 93% identical to VIM-2) revealed significant differences in K(m) values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Molecular modelling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behaviour.
Docquier
Jean-Denis
JD
Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, Policlinico 'Le Scotte', Viale Bracci, I-53100 Siena, Italy.
Lamotte-Brasseur
Josette
J
Galleni
Moreno
M
Amicosante
Gianfranco
G
Frère
Jean-Marie
JM
Rossolini
Gian Maria
GM
D000595:Amino Acid Sequence; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006868:Hydrolysis; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
D002614:Chelating Agents; D003470:Culture Media; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C417264:VIM-1 metallo-beta-lactamase; D001618:beta-Lactamases