Twelve Positions in a β-Lactamase That Can Expand Its Substrate Spectrum with a Single Amino Acid Substitution (original) (raw)
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Molecular Microbiology, 1994
In order to understand how TEM-1 p-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidlme was determined. It was found that the sequence requirements for wild-type leveis of ampiciilin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region resuit in levels of ceftazidime resistance at ieast three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-foid increase in ceftazidime resistance were identified. Characterization of altered p-lactamase enzymes indicated that while their catalytic efficiency (fccat/'^m) for ceftazidime hydrolysis is higher, the enzymes are pooriy expressed relative to wild-type TEM-1 p-lactamase.
Journal of Biological Chemistry, 1999
We investigated the effects of mutations at positions 164 and 179 of the TEM pUC19 -lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum -lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A -lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum -lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg 164 3 Asn, Arg 164 3 Ser, Asp 179 3 Asn, and Asp 179 3 Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum -lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K s) invariably increased. Computation of the microscopic rate constants (k 2 and k 3) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k 3) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k 2) and deacylation (k 3) of the extended-spectrum -lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM pUC19 -lactamase. Our results indicated that severe steric interactions between the large 7 functionalities of the expanded-spectrum -lactams and the ⍀-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity. Production of -lactamases is the most common mechanism of high-level resistance to -lactam antibiotics in bacteria.
Scientific reports, 2016
The omega loop in β-lactamases plays a pivotal role in substrate recognition and catalysis, and some mutations in this loop affect the adaptability of the enzymes to new antibiotics. Various mutations, including substitutions, deletions, and intragenic duplications resulting in tandem repeats (TRs), have been associated with β-lactamase substrate spectrum extension. TRs are unique among the mutations as they cause severe structural perturbations in the enzymes. We explored the process by which TRs are accommodated in order to test the adaptability of the omega loop. Structures of the mutant enzymes showed that the extra amino acid residues in the omega loop were freed outward from the enzyme, thereby maintaining the overall enzyme integrity. This structural adjustment was accompanied by disruptions of the internal α-helix and hydrogen bonds that originally maintained the conformation of the omega loop and the active site. Consequently, the mutant enzymes had a relaxed binding cavity...
Evolution of an Antibiotic Resistance Enzyme Constrained by Stability and Activity Trade-offs
Journal of Molecular Biology, 2002
Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the b-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182 ! Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.
Molecular Microbiology, 1990
Gram-negative bacteria. The two ubiquitous types of enzyme have a large spectrum of activity and preferentially hydrolyse the penicillins as well as some first-and second-generation cephalosporins. Recently, point mutations in the corresponding genes have been observed, apparently selected for, in the clinical setting, by originally '|3-lactamasestable' third-generation cephalosporins or by monobactams, vi/hich fall into the substrate range of the mutant or 'extended-spectrum' |j-lactamases. The point mutations are clustered in three areas, each adjacent to one of the seven evolutionarily conserved boxes described by Joris etal. (1988). The substituted amino acids at positions 102 (adjacent to the iv-3 helix), 162 (adjacent to the i«-7 helix) and 235,236 and 237 (on the ii-3 strand) are located in close proximity to the active-site cavity and are thought to open up novel enzyme-substrate interactions, involving, in particular, the oxyimino moieties of the newer ;3-lactam compounds.
PLoS Computational Biology, 2013
The bacterial enzyme b-lactamase hydrolyzes the b-lactam ring of penicillin and chemically related antibiotics, rendering them ineffective. Due to rampant antibiotic overuse, the enzyme is evolving new resistance activities at an alarming rate. Related, the enzyme's global physiochemical properties exhibit various amounts of conservation and variability across the family. To that end, we characterize the extent of property conservation within twelve different class-A b-lactamases, and conclusively establish that the systematic variations therein parallel their evolutionary history. Large and systematic differences within electrostatic potential maps and pairwise residue-to-residue couplings are observed across the protein, which robustly reflect phylogenetic outgroups. Other properties are more conserved (such as residue pK a values, electrostatic networks, and backbone flexibility), yet they also have systematic variations that parallel the phylogeny in a statistically significant way. Similarly, the above properties also parallel the environmental condition of the bacteria they are from in a statistically significant way. However, it is interesting and surprising that the only one of the global properties (protein charge) parallels the functional specificity patterns; meaning antibiotic resistance activities are not significantly constraining the global physiochemical properties. Rather, extended spectrum activities can emerge from the background of nearly any set of electrostatic and dynamic properties.
Prediction of the Evolution of Ceftazidime Resistance in Extended-Spectrum -Lactamase CTX-M-9
Antimicrobial Agents and Chemotherapy, 2006
A random mutagenesis technique was used to predict the evolutionary potential of -lactamase CTX-M-9 toward the acquisition of improved catalytic activity against ceftazidime. Thirty CTX-M mutants were obtained during three rounds of mutagenesis. These mutants conferred 1-to 128-fold-higher MICs of ceftazidime than the parental enzyme CTX-M-9. The CTX-M mutants contained one to six amino acid substitutions. Mutants harbored the substitutions Asp240Gly and Pro167Ser, which were previously observed in clinical CTX-M enzymes. Additional substitutions, notably Arg164His, Asp179Gly, and Arg276Ser, were observed near the active site. The kinetic constants of the three most active mutants revealed two distinct ways of improving catalytic efficiency against ceftazidime. One enzyme had a 17-fold-higher k cat value than CTX-M-9 against ceftazidime. The other two had 75-to 300-fold-lower K m values than CTX-M-9 against ceftazidime. The current emergence of CTX-M -lactamases with improved activity against ceftazidime may therefore be the beginning of an evolutionary process which might subsequently generate a great diversity of CTX-M-type ceftazidimases.
Antimicrobial Agents and Chemotherapy, 2015
Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli . Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant β-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the ...
Antimicrobial agents and chemotherapy, 2017
The unwelcome evolution of resistance to the advanced generation cephalosporin antibiotic, ceftazidime is hindering the effective therapy of Burkholderia cepacia complex (BCC) infections. Regrettably, BCC organisms are highly resistant to most antibiotics, including polymyxins; ceftazidime and trimethoprim-sulfamethoxazole are the most effective treatment options. Unfortunately, resistance to ceftazidime is increasing and posing a health threat to populations susceptible to BCC infection. We found that up to 36% of 146 tested BCC clinical isolates were nonsusceptible to ceftazidime (MICs ≥ 8 μg/ml). To date, the biochemical basis for ceftazidime resistance in BCC is largely undefined. In this study, we investigated the role of the Ω-loop in mediating ceftazidime resistance in the PenA β-lactamase from Burkholderia multivorans, a species within the BCC. Single amino acid substitutions were engineered at selected positions (R164, T167, L169, and D179) in the PenA β-lactamase. Cell-bas...