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Complete amino acid sequence of p453-plasmid-mediated PIT-2 beta-lactamase (SHV-1).

ABSTRACT: The complete amino acid sequence of the p453-plasmid-mediated PIT-2 beta-lactamase (SHV-1) was determined. The protein contains 265 residues. Peptides resulting from digestions with trypsin, Staphylococcus aureus V8 proteinase, chymotrypsin and Lys-C proteinase and cleavage with CNBr were separated and purified by using reverse-phase h.p.l.c. The amino acid sequence of each peptide was manually determined with the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method. The primary structure of PIT-2 beta-lactamase was compared with those of two closely related enzymes, namely TEM-1 beta-lactamase and the beta-lactamase of Klebsiella pneumoniae strain LEN-1. The PIT-2 beta-lactamase amino acid sequence was strongly retained, with respectively 68% and 88% homology. Thus PIT-2 enzyme could represent an evolutionary step between a chromosomally encoded beta-lactamase and the plasmid-mediated TEM beta-lactamases.

SUBMITTER: Barthelemy M

PROVIDER: S-EPMC1148965 | biostudies-other | 1988 Apr

REPOSITORIES: biostudies-other

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Project description:Beta-lactamases are enzymes that catalyze the hydrolysis of beta-lactam antibiotics. beta-lactamase/beta-lactamase inhibitor protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. BLIP is a 165 amino acid protein that inhibits several class A beta-lactamases with a wide range of affinities: picomolar affinity for K1; nanomolar affinity for TEM-1, SME-1, and BlaI; but only micromolar affinity for SHV-1 beta-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high-resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of computational design methodology. We used EGAD, a physics-based computational design program, to redesign BLIP in an attempt to increase affinity for SHV-1. Characterization of several of designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10- to 1000-fold relative to wild type BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of the calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Structures of the two complexes with the highest affinity, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 A resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in the experimental structures, but not in the predicted design conformations. This discrepancy highlights the difficulty of modeling salt bridge interactions with a protein design algorithm that approximates side chains as discrete rotamers. Nevertheless, while local structural features of the interface were sometimes miscalculated, EGAD is globally successful in designing complexes with increased affinity.

Project description:Among the TEM-type extended-spectrum beta-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV beta-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in beta-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys beta-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser beta-lactamases. Correspondingly, the overall catalytic efficiency (kcat/Km) of hydrolysis for cefotaxime was increased from 0.60 +/- 0.07 microM(-1) s(-1) (mean +/- standard deviation) for Gly238Ser to 1.70 +/- 0.01 microM(-1) s(-1) for the Asp104Lys and Gly238Ser beta-lactamase (threefold increase). We also showed that (i) k3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser beta-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 beta-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.

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Complete amino acid sequence of p453-plasmid-mediated PIT-2 beta-lactamase (SHV-1).

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