Abstract
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, allowing for access of new substrates, which regrouped upon substrate binding in an induced-fit manner for subsequent hydrolytic reactions. Together, the data demonstrate that the design of the binding cavity, including the omega loop with its enormous adaptive capacity, is the foundation of the continuous evolution of β-lactamases against new drugs.
Highlights
The structural basis of the mechanism underlying the highly adaptive capacity of the omega loop, which leads to the evolution of β-lactamases that can hydrolyze new drugs by providing a flexible active site
We obtained TR mutations of penL, which codes for a class A β-lactamase, by exposing Burkholderia thailandensis strain E26424 to ceftazidime (3–5 μg/ml), a third-generation cephalosporin
PenL has been used as a model in exploring various evolutionary paths to substrate spectrum extension through amino acid substitutions, deletions, and duplications[4,10,20,21]
Summary
TR mutations in the omega loop-coding region in the penL gene. We obtained TR mutations of penL (penA in our previous reports[4,10,20,21] is renamed penL here, following the nomenclature guidelines by Poirel et al.23), which codes for a class A β-lactamase, by exposing Burkholderia thailandensis strain E26424 to ceftazidime (3–5 μg/ml), a third-generation cephalosporin. Similar to other class A β-lactamases, the active site cavity of PenL-WT can be depicted as a tetrahedral organization, where one plane is open for the substrates, while the other three are composed of the omega loop (residues 161–179), structures including the β3 strand (residues 230–240), and the 102–107 loop and the neighboring 130– 132 bend (Fig. 3A) These three planes meet at the center, where the N-terminal end of the α2 helix, containing the catalytic Ser[70], is located (Fig. 3A). In the PenL-tTR structures, perturbations in the omega loop resulted in the dislocation of a number of nearby residues, along with the disorganization of the α-helix in the omega loop (Glu166-Asn170), which most notably led to the breakage of a salt bridge that is normally present between Thr[167] and Arg[104] and tightly packs the active-site cavity (Fig. 4) These disruptions further led to the dislocation of the 102–107 loop; the neighboring α3, α4, α5 helices; and Asp[240], which is in the C-terminal section of the β3 strand (Fig. 5). The restoration of the α-helix was not observed in PenL-tTR11-CBA, likely due to the longer tTR11 causing higher disorders in the region
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