Specificity determinants of the class A [beta]-lactamase RTEM-1

Citation

Hollenbaugh, Diane (1991) Specificity determinants of the class A [beta]-lactamase RTEM-1. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/0qza-et62. https://resolver.caltech.edu/CaltechETD:etd-06192007-142753

Abstract

Factors determining the substrate specificity of RTEM-1 [beta]-lactamase have been studied with the use of cassette mutagenesis. Three studies were performed to elucidate these factors. First, two chimeric proteins were created, substituting the active site helix of RTEM-1, residues 71-82, with the corresponding sequence from PBP5 of E. coli. In Chimera2, the potential disulfide bond between Cys77 and Cys123 was preserved, whereas it was removed in Chimera1 by a C77S mutation. The expression of either chimera produced no observable phenotypic effects. Both chimeras were present in E. coli only at reduced temperatures (25°C). Chimera2 was more stable to periplasmic proteases than Chimera1, suggesting that the disulfide bond was formed, increasing the stability. The formation of the disulfide was verified by an SDS-PAGE mobility shift assay. The behavior of Chimera2 and RTEM-1 on ion-exchange and chromatofocusing columns and the circular dichroism (CD) spectra of the two enzymes suggested a significant structural difference. Chimera2 was much more sensitive to thermal denaturation than RTEM-1, as monitored by CD. Chimera2 displayed no detectable activity as a [beta]-lactamase against benzylpenicillin or as a depsipeptidase against diacetyl-Lys-D-Ala-D-lactate in assays with detection limits of 3 x 10(-3) sec(-1) and 3 x 10(-3) sec(-1), respectively. Radiolabelled acyl-enzyme could not be detected following incubation of Chimera2 with [[superscript 14] C]-benzylpenicillin.

In the second study, residues 233, 234, 235 and 237 of RTEM-1 [beta]-lactamase were substituted with residues believed to be important in the differing substrate specificities of the penicillin-recognizing enzymes. Mutants were generated to yield all possible combinations of the substitutions D233H, K234H, K234N, K234Q, S235T and A237N, resulting in thirty-two sequences, including the wild-type sequence. Results of phenotypic screening were as follows: 1) Mutants with a substitution at Lys234 were phenotypically inactive against all antibiotics tested. 2) The substitution D233H destabilized the enzyme as seen by differences in the conferred resistance at 30°C vs. 37°C. 3) The substitution S235T led to increased resistance to cephalothin compared to RTEM-1. Molecular modeling studies suggest that the instability imparted by the D233H substitution may be due to the accommodation of the five-membered ring of histidine and the loss of a favorable contact between Asp233 and Arg222. These studies also suggest that the incorporation of the methyl group in the S235T mutants into the packed space within the structure alters the orientation of the hydroxyl group. This hydroxyl group is hydrogen-bonded to the ammonium group of the conserved Lys234, which has been shown previously to be necessary in the binding of the substrate.

In the third study of RTEM-1 substrate specificity, residues 237, 238 and 240, were simultaneously randomized, and mutants were screened for the ability to confer resistance to the antibiotic cefotaxime. The three residues were chosen for study based on the sequences of the naturally occurring extended-spectrum [beta]-lactamases, which are active against the third generation cephems. Sixty-eight different mutant enzymes were found to possess extended activity. In general, small amino acids were found at position 237 (Ser, Gly, Ala, Thr), a range of residues were found at position 238 (Ala, Asn, Arg, Ser, Thr, Asp, Gly), and a positively charged residue (Lys, Arg, His) was commonly found at position 240. Glycine was found at position 238 if and only if proline was present in position 240. All of the mutants conferred a reduced level of resistance to ampicillin and benzylpenicillin compared to the wild-type RTEM-1. The majority of the mutants conferred increased resistance to cephalothin and cloxacillin. The values of kcat and KM, were determined for three of the mutants, ASK, ASE and AAK. The values of kcat on the substrate benzylpenicillin were only slightly reduced from the values obtained for the wild-type RTEM-1 enzyme. The values of KM for both benzylpenicillin and cefotaxime were reduced substantially, with the value of KM on cefotaxime for the most active mutant, ASK, reduced to 120µM, compared with Ki of > 3 mM for the wild-type RTEM-1. With a consideration of the structural differences between benzylpenicillin and cefotaxime and the results of molecular modeling studies, the results suggest that the effect of these mutations on the substrate specificity of the enzyme is not a simple enzyme-substrate contact as was previously proposed but instead suggest that the substitutions allow a conformational change not accessible to the wild-type enzyme.

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