Analysis of the molecular relatedness of four extended spectrum beta-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5) by comparative protein titration curves.
Six beta-lactamases from Klebsiella pneumoniae, five of which (SHV-1, SHV-2, SHV-3, SHV-4 and SHV-5) were plasmid-encoded and one which (beta 1a GN 11-03) was chromosomally-encoded, were compared by analysis of their isoelectric points (pI), electrophoresis mobilities (MF) and titration curves or pH gradient electrophoresis. Four groups were defined by their pI and MF, namely SHV-1 and SHV-2 (pI = 7.6, MF approximately 14), SHV-3 and beta 1a GN 11-03 (pI = 7.0, MF approximately 20), SHV-4 (pI = 7.8 MF approximately 12) and SHV-5 (pI = 8.2, MF approximately 5). The titration curves of SHV-1 and SHV-2 enzymes on the one hand, and SHV-3 and beta 1a GN 11-03 on the other were completely superimposable for the whole of the pH gradient (3.5-10), indicating strongly similarity. Conservative amino-acid substitutions could account for the differences in the substrate spectra of the purified enzymes. The differences observed between the titration curves of the enzymes SHV-1/SHV-3, SHV-1/beta 1a GN 11-03, SHV-2/SHV-3 and SHV-5/SHV-4 pairs were consistent with the replacement of a basic amino-acid residue in the former enzyme of each pair by an acidic residue in the latter. Similarly, the titration curves of SHV-1/SHV-4 and of SHV-2/SHV-4 pairs may suggest the replacement of an acidic amino-acid in the former beta-lactamases by a neutral amino-acid in the latter of each pair. However, the presence of several self-cancelling or neutral substitutions is also possible. In contrast, when SHV-1 and TEM-1 (pI = 5.4 MF approximately 45) were titrated together, no structural relationship could be inferred.
Vedel
G
G
Laboratoire de Bactériologie, CHU Cochin, Paris, France.
Picard
B
B
Paul
G
G
Philippon
A
A
Gilly
L
L
Krishnamoorthy
R
R
Névot
P
P
D002876:Chromosomes, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics
D001618:beta-Lactamases