Substitution of lysine at position 104 or 240 of TEM-1pTZ18R .beta.-lactamase enhances the effect of serine-164 substitution on hydrolysis or affinity for cephalosporins and the monobactam aztreonam

Abstract

By site-directed mutagenesis, TEM-1 beta-lactamase was altered to contain single amino acid changes of E104K, R164S, and E240K, in addition to double changes of E104K/R164S or R164S/E240K and the triple change of E104K/R164S/E240K. Hydrolysis rates for cephaloridine and benzylpenicillin were lowered at least 1 order of magnitude for all enzymes containing R164S substitutions. All mutant enzymes exhibited increased kcat values for beta-lactam antibiotics containing an aminothiazole oxime side chain. Hydrolysis of ceftazidime was most affected, with kcat values increased 3-4 orders of magnitude in all enzymes with the substituted R164S moiety. Km values decreased for all substrates except ceftazidime in the enzymes with multiple mutations. Aztreonam was most affected, with Km values lowered 23-56-fold in the enzymes bearing multiple mutations. When the crystal structures of aztreonam and related monobactams were studied and projected into an active-site model of the PC1 beta-lactamase, it became apparent that the two lysine residues might serve equivalent roles by interacting with the carboxylate of the aminothiazole oxime side chain. Hydrogen-bonding interactions involving the oxime and N7 of the lysine, particularly Lys-104, may also be important in some antibiotics. Ser-164 apparently serves an indirect role, since it is somewhat distant from the active-site cleft.

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases

“…In general, our results with the mutations Lys-104, Ser-164, and Lys-240 are in accordance with those reported previously (28,30). Sowek et al (30) found a catalytic efficiency for cephaloridine in the Ser-164 mutant that was higher than ours, which can be related to the differences in the studied wild-type bla gene and/or in the enzyme preparation.…”

“…Sowek et al (30) found a catalytic efficiency for cephaloridine in the Ser-164 mutant that was higher than ours, which can be related to the differences in the studied wild-type bla gene and/or in the enzyme preparation. These three mutations, particularly Lys-104 and Ser-164, are in fact more frequently found among the more active extended-spectrum ␤-lactamases.…”

“…Nevertheless, some of those mutants were constructed in different plasmids or genetic contexts. Indeed, the bla genes used for some of those constructions were not the original bla TEM-1 genes but derivatives with theoretically ''neutral'' mutations (11,30). The aim of this work was to construct a collection of TEM-1 derivatives in the same genetic context (plasmid, promoter, or strain) and define the change in the enzyme properties (kinetics, amount, and stability) and in the phenotype conferred by each one of these mutants.…”

“…These changes at positions 104 and 240 consistently decreased susceptibility to ceftazidime and aztreonam. It has been sugested that enzymes with lysines at positions 104 and 240 could establish electrostatic interactions with the oxime acid group of ceftazidime and aztreonam (30).…”

“…For each position, the replacement is with a particular amino acid, the only exception being Arg-164, which can be replaced by either Ser or His. Several authors constructed TEM-1 derivatives containing some of the abovedescribed single mutations by either oligonucleotide-directed mutagenesis (10,30) or subcloning of fragments containing those mutations (28). Biochemical and microbiological studies have been performed with some of these mutants.…”