Combinatorial biochemistry and shuffling of TEM, SHV and Streptomyces albus omega loops in PSE-4 class A ß-lactamase

François Sanschagrina, Esther Thériaulta, Yves Sabbagha, Normand Voyerb and Roger C. Levesquea,*

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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The class A PSE-4 ß-lactamase was used for studying the importance of amino acids in the omega ({Omega}) loop and its interactions for hydrolysis of ß-lactam antibiotics. By cassette mutagenesis, we replaced the amino acids 163–179 {Omega} loop in PSE-4 with TEM-1, SHV-1 and Streptomyces albus G ß-lactamase {Omega} loops. Phenotypic analysis of Escherichia coli recombinants expressing the {Omega} loop PSE-4 mutant enzymes gave MICs and kinetic data similar to those of wild-type PSE-4.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The omega () loop region is known from studies of the TEM-1 structure to be implicated in substrate specificity. The roles of amino acids 161–179 in modulating ceftazidime resistance and hydrolysis in the PSE-4 loop are different from those in TEM-1.1

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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Escherichia coli JM101 (supE, thi ({triangleup}lac-proAB), F', traD36, proAB, lacIqZDM15) was the recipient strain for construction and production of single strand DNA for mutagenesis and sequencing. E. coli DH5{alpha} (supE44 {triangleup}lacU169 ({phi}80, lacZ{triangleup}M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was the recipient strain for ß-lactamase plasmids and MIC determinations. All proteins were expressed in E. coli BL21({lambda}DE3 F ompT rb mb) strain (Novagen, Madison, WI, USA). The plasmid pMON711 containing blaPSE-4 was used to perform cassette mutagenesis as previously described.3 Preparation of DNA and related techniques are standard methods.4 The replacements of the blaPSE-4 nucleotide sequences coding from D163 to T179 with the TEM, SHV and S. albus G loops was performed by ligation of each respective loop DNA fragment into pMON711 previously digested with XbaI and Bsu36I enzymes and dephosphorylated. The synthetic loops contained XbaI–Bsu36I cohesive ends prepared by annealing of two complementary oligonucleotides.5 Three different fragments were constructed as complementary DNA strands using six oligonucleotides to create the TEM-1, SHV-1 and S. albus G ß-lactamase loops. Screening of mutants was performed by nucleotide sequencing. The structural genes of selected clones containing blaPSE-4 with desired mutations were completely sequenced. Minimum inhibitory concentrations (MICs), ß-lactamase purifications and determination of kinetic constants were achieved as previously described.3


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In general, the differences in MICs (Table IGo) between E. coli cells expressing wild-type PSE-4 enzyme and the three PSE-4 loop mutants represent one dilution (two-fold), which cannot be considered significant.


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Table I. Phenotypic analysis (MIC) of E. coli DH5{alpha} carrying PSE-4 and mutant loop ß-lactamases
 
There were no significant variations in the level of expression for the mutant enzymes compared with wild-type PSE-4, as revealed by Western transfer experiments using an anti-PSE-4 polyclonal antibody (data not shown).

The kinetic parameters for the wild-type PSE-4 and the PSE-4 loop mutants are shown in Table IIGo. In general, the differences between PSE-4 mutant enzymes and the wild-type show four-fold variation, which cannot be considered as significant.


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Table II. Kinetic parameters (mean ± s.d. from triplicates) obtained for PSE-4 and mutant loop ß-lactamases with ampicillin and carbenicillin
 
The data obtained by circular dichroism indicated no significant differences in the global secondary structures of PSE-4 loop mutants compared with wild-type PSE-4 (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we wanted to determine whether the PSE-4 loop could be replaced by homologous loop amino acid sequences from TEM, SHV and S. albus enzymes. We anticipated that these loops from other class A ß- lactamases would presumably maintain hydrolysis without greatly impinging on hydrolytic activity against ß-lactams. At the same time, we expected that such enzymatic combinatorial biochemistry could define the roles of specific amino acids in the PSE-4 loop for preferential hydrolysis of specific substrates such as carbenicillin compared with ampicillin. As expected, all ß-lactamase mutants conserved high levels of resistance against penicillins. Minimum inhibitory concentrations of carbenicillin for loop mutants were not significantly different from those of wild-type PSE-4. Differences in the kinetic parameters of carbenicillin were not sufficient to affect activity against all mutants. Altogether, these results showed that the loop is not involved in the substrate specificity of carbenicillin in PSE-4 and support the random replacement mutagenesis data of PSE-4 loop.1

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
 
The Medical Research Council of Canada supported this work as a grant to R.C.L.


    Notes
 
* Corresponding author. Tel: +1-418-656-3070; Fax: +1-418-656-7176; E-mail: rclevesq{at}rsvs.ulaval.ca. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Therrien, C., Sanschagrin, F., Palzkill, T. & Levesque, R. C. (1998). Roles of amino acids 161 to 179 in the PSE-4 omega loop in substrate specificity and in resistance to ceftazidime. Antimicrobial Agents and Chemotherapy 42, 2576–83.[Abstract/Free Full Text]

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, 1225–30.[Abstract/Free Full Text]

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, 2319–25.[Abstract/Free Full Text]

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, 10482–9.[Abstract/Free Full Text]

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, 939–46.[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, 3286–96.[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, 671–8.[ISI][Medline]

Received 1 July 1999; returned 22 September 1999; revised 15 October 1999; accepted 26 October 1999





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