Random replacement mutagenesis was used to identify amino acids in the Ω loop that could modulate substrate specificity and to examine the effect of amino acid substitutions on the level of PSE-4 expression. Screening of the six Ω loop libraries with high concentrations (1 mg/ml) of ampicillin or carbenicillin yielded differences in the percentage of functional β-lactamases. The 162–164 and 165–167 libraries gave a 10-fold-higher percentage of functional amino acid sequences with the carbenicillin selection compared with the ampicillin selection. This finding indicated that the sequence requirements for a wild-type level of resistance toward both penicillins were more stringent for ampicillin than for carbenicillin at these positions. This particularity seemed to apply to specific regions of the Ω loop, especially the 162 to 164 and 165 to 167 regions. Furthermore, the selective pressure exerted by 10 μg of ampicillin per ml was equivalent to a 100-fold-higher concentration of carbenicillin. With these results, we hypothesized that ampicillin hydrolysis was affected more by substitutions and that specific interactions must be present to maintain efficient hydrolysis. In contrast, carbenicillin hydrolysis required fewer specific interactions with amino acid residues in the Ω loop and possibly other residues near or in the active site. Sequence analysis of functional mutants selected with ampicillin or with carbenicillin identified four invariant residues, R164, E166, R178, and D179, which are critical for ampicillin or carbenicillin hydrolysis. N170 and D176 also appeared to be important for a wild-type level of function. These residues were previously identified from high-resolution X-ray structures (10
) and site-directed mutagenesis studies (1
) to be implicated in the catalytic activity and active-site conformation of β-lactamases.
Contrasting of the sequence requirements (e.g., amino acid sequences) for function with high concentrations of ampicillin or carbenicillin did not permit the identification of a single amino acid in the Ω loop that could interact specifically with either one of the penicillins studied. The small number of mutants sequenced for each selection could explain the lack of correlation between the percentage of functional amino acid sequences and the sequence requirements for function. Furthermore, the MICs of ampicillin and carbenicillin for the mutants isolated with high concentrations of either penicillin were affected in a similar manner by the substitution; this was observed for the majority of mutants. There appears to be no major determinant of specificity in the Ω loop of PSE-4 that could discriminate for the preferential hydrolysis of ampicillin over carbenicillin.
To investigate the potential of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we screened the six Ω loop libraries with ceftazidime at a concentration of 0.5 μg/ml. This concentration restrained the growth of E. coli cells expressing wild-type PSE-4. Thus, potential screens were considered to have an extended-spectrum profile and were assumed to be able to hydrolyze ceftazidime more efficiently. Functional amino acid sequences consistent with a higher-than-wild-type level of resistance toward ceftazidime were identified in a precise region of the Ω loop. In contrast to TEM-1 β-lactamase, the structural modification in the 162–164 and 174–176 regions of PSE-4 by amino acid substitutions was not sufficient to increase the level of ceftazidime hydrolysis.
The structural basis for this observation is difficult to explain without crystallographic data for PSE-4. The amino acids implicated in salt bridge formation in the TEM-1 Ω loop (K73: E166, R161: D163, R164: E171, R164: D179, and D176: R178) (12
) are conserved in PSE-4 (Fig. ). From this homology, it was predicted that similar interactions would be present in the PSE-4 Ω loop; removal of these structural constraints would allow the entry of the bulky side chain of ceftazidime, as was postulated for TEM-1 ceftazidime mutants (19
). Two tentative explanations could be envisaged: first, the conformation and the network of interactions between the amino acids of the Ω loop of PSE-4 are different from those of TEM-1. Second, the need for specific interactions with ceftazidime is required for efficient hydrolysis. An additional piece of data supporting the structural differences in the Ω loop between PSE-4 and TEM is the lower level of expression for PSE-4 mutants selected on ampicillin, while TEM-1 ampicillin mutants were found to be expressed at levels similar to that of the native enzyme (19
). Thus, it is possible that other types of amino acid interactions are as important as the putative salt bridge interaction in the stabilization of the PSE-4 structure. The sequence requirements for a stably expressed protein are more stringent for PSE-4 than for TEM-1. Low levels of protein expression resulting from a single or multiple mutations could be due to many factors, such as thermodynamic alterations, altered folding kinetics, or increased protease susceptibility resulting from amino acids exposed to the solvent. Since a correlation between enzyme susceptibility to proteases and thermal stability exists (20
), it is convenient to think that such phenomenon explains the observations with the majority of the Ω loop PSE-4 mutations. Further experiments measuring protein stability will be essential to identify the exact causes of low levels of expression in these PSE-4 mutants.
Sequence alignment of amino acids from the PSE-4 and TEM-1 Ω loops.
Sequence requirements for clinically relevant levels of ceftazidime resistance (16 to 32 μg/ml) were found to be stringent in some TEM-1 mutants. These functional sequences were only found in the 165 to 167 region. Determinants A, Y or F165, Y or H166, and G167 only have been described previously (19
). Such specificity determinants were observed with ceftazidime mutants selected from the 165–167 library; a G167 was found in two mutants, while a Y was found at position 165. However, these new observations are quite unique and merit further investigations to determine if these changes have the same effect in other class A enzymes.
The catalytic activity of mutants selected on ampicillin and carbenicillin was affected more when changes were in specific regions of the Ω loop, especially the 162 to 164, 168 to 170, and 171 to 173 positions. We specify that 100% of wild-type β-lactamase activity was not a prerequisite for high levels of ampicillin and carbenicillin resistance. Since a direct correlation exists between the amount of enzyme and the level of activity, the decreased in vitro activity seen for the majority of mutants was due to the low level of β-lactamase expression. Kinetic analysis of the 162MDR164 mutant revealed that the substitution enhanced the apparent affinity and reduced the catalytic activity of the enzyme. The relief of critical structural interactions in the Ω loop could disturb the correct positioning of chemical groups involved in the catalysis of substrates. This hypothesis was introduced previously in the random replacement mutagenesis studies of TEM-1 derivatives that were able to hydrolyze ceftazidime and that showed no activity toward the preferred substrate (19
). Further evidence to support this hypothesis comes from structural data extracted from the crystal of the P54 β-lactamase mutant of Staphylococcus aureus
). It was found that the elimination of the salt bridge between R164 and D179 by substituting for the latter with an N substantially disordered the Ω loop and resulted in a drastic decrease in activity. It was also found that deacylation of the acyl-enzyme complex of penicillin G and P54 enzyme was the rate-limiting step. Moreover, kinetic analysis of cefepime hydrolysis by the D179G and R164N mutant variants of TEM-1 β-lactamase showed lowered values for the dissociation constants Ks
for both mutants (25
). Circular dichroic analysis of the R164N enzyme indicated a decrease in helicity for this mutant compared to the native structure. The structural modifications of the active site were proposed to accommodate the kinetic and the structural data (25
In conclusion, the data obtained from this study confirmed the important roles of R164, E166, N170, D176, R178, and D179 in the structure and function of PSE-4. Sequence requirements of the Ω loop consistent with a stably expressed protein were more stringent for PSE-4 than for TEM-1. The determinants of carbenicillin specificity were not found in the Ω loop region of PSE-4, and, finally, the mechanism responsible for substrate specificity toward expanded-spectrum cephalosporins of PSE-4 seemed to be quite different from that of TEM-1.