a Microbiologie Moléculaire & Génie des Protéines, Sciences de la Vie et de la Santé, Faculté de Médecine, Pavillon Charles-Eugène Marchand, Université Laval; b Département de Chimie, Pavillon Alexandre-Vachon, Université Laval, Ste-Foy, Québec, Canada G1K 7P4
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As a model for class A enzymes capable of hydrolysing carbenicillin at a high rate, we used the plasmid-mediated prototype PSE-4 ß-lactamase from Pseudomonas aeruginosa strain Dalgleish.2 We present data to attempt to define the role of loops in class A ß-lactamases by replacing the PSE-4
loop with those from TEM-1, SHV-1 and Streptomyces albus enzymes using a cassette mutagenesis method. The results of these experiments are supported by phenotypic and biochemical analysis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The kinetic parameters for the wild-type PSE-4 and the PSE-4 loop mutants are shown in Table II
. In general, the differences between PSE-4 mutant enzymes and the wild-type show four-fold variation, which cannot be considered as significant.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo resistance to ampicillin decreased two-fold for loop mutants; these results agreed with catalytic efficiencies, which also decreased between two- and four-fold for PSE-4 mutant enzymes. All mutants had an increase in Km and no significant changes in kcat. We postulate that of the I165W, N168Q, G172A and L174P substitutions, maybe one, several or all changes could be responsible for the increase of Km with ampicillin, because all these substitutions were present in all mutants. Position 165 was identified among invariant residues responsible for ampicillin or carbenicillin hydrolysis in PSE-4.1 Piperacillin has a large side-chain like oxacillin but substitutions in the
loop did not affect MICs for piperacillin as with oxacillin; this result can be explained only by the fact that the
loop does not play a role in substrate specificity for hydrolysis of piperacillin in PSE-4.
The lower MIC values with oxacillin for PSE-4 and PSE-4 SHV compared with those of PSE-4
TEM and PSE-4
S. albus G can be explained by similar residues common in PSE-4
TEM and PSE-4
S. albus G and not found in PSE-4
SHV and wild-type PSE-4. There are two P167 and P174 residues in PSE-4
TEM and in PSE-4
S. albus G. We suggest that P167T substitution in PSE-4
SHV could modify interactions with oxacillin and explain the decreasing levels of resistance. The presence of two proline residues in the PSE-4
TEM and PSE-4
S. albus G mutants could modify the topology of the active site and enhance the hydrolytic activity of PSE-4
TEM and PSE-4
S. albus G for oxacillin; a situation reminiscent of TEM-1 with ceftazidime.6
The levels of resistance to cephaloridine and cefoperazone were not greatly modified, suggesting that none of the loop shufflings was important for changes in hydrolysis of these antibiotics.
The loop replacement experiments done in PSE-4 demonstrated that even if the PSE-4 active site is significantly different from TEM-1 and other ß-lactamases,1,3,7 the
loop replacements can be made without significant changes in hydrolytic activity towards ß-lactams. Based upon these results, the
loop deletions8 and replacements which demonstrated the importance of E166 in the deacylation step,9 the structural role of the PSE-4
loop is to position correctly the residue E166 for the deacylation step.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Boissinot, M. & Levesque, R. C. (1990). Nucleotide sequence of the PSE-4 carbenicillinase gene and correlations with the Staphylococcus aureus PC1 ß-lactamase crystal structure. Journal of Biological Chemistry 265, 122530.
3
.
Sabbagh, Y., Theriault, E., Sanschagrin, F., Voyer, N., Palzkill, T. & Levesque, R. C. (1998). Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: importance of residues 216 to 218 in class A ß-lactamases. Antimicrobial Agents and Chemotherapy 42, 231925.
4 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY.
5 . Richards, J. H. (1991). In Directed Mutagenesis: A Practical Approach, (McPherson, M. J., Ed.). IRL Press, London.
6
.
Maveyraud, L., Saves, I., Burlet-Schiltz, O., Swaren, P., Masson, J. M., Delaire, M. et al. (1996). Structural basis of extended spectrum TEM ß-lactamases. Crystallographic, kinetic, and mass spectrometric investigations of enzyme mutants. Journal of Biological Chemistry 271, 104829.
7 . Lenfant, F., Petit, A., Labia, R., Maveyraud, L., Samama, J. P. & Masson, J. M. (1993). Site-directed mutagenesis of ß-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes. European Journal of Biochemistry 217, 93946.[Abstract]
8 . Banerjee, S., Pieper, U., Kapadia, G., Pannell, L. K. & Herzberg, O. (1998). Role of the omega-loop in the activity, substrate specificity, and structure of class A ß-lactamase. Biochemistry 37, 328696.[ISI][Medline]
9 . Leung, Y. C., Robinson, C. V., Aplin, R. T. & Waley, S. G. (1994). Site-directed mutagenesis of ß-lactamase I: role of Glu-166. Biochemical Journal 299, 6718.[ISI][Medline]
Received 1 July 1999; returned 22 September 1999; revised 15 October 1999; accepted 26 October 1999