Increased Structural Flexibility at the Active Site of a Fluorophore-conjugated β-Lactamase Distinctively Impacts Its Binding toward Diverse Cephalosporin Antibiotics

Abstract

The ⍀-loop at the active site of ␤-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the ⍀-loop has been a general strategy for molecular evolution of ␤-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the ⍀-loop. Yet no structural information is available that demonstrates such flexibility or its relation to substrate profile and enzyme kinetics. Here we report an engineered ␤-lactamase that contains an environment-sensitive fluorophore conjugated near its active site to probe the structural dynamics of the ⍀-loop and to detect the binding of diverse substrates. Our results show that this engineered ␤-lactamase has improved binding kinetics and positive fluorescence signal toward oxyimino-cephalosporins, but shows little such effect to non-oxyimino-cephalosporins. Structural studies reveal that the ⍀-loop adopts a less stabilized structure, and readily undergoes conformational change to accommodate the binding of bulky oxyimino-cephalosporins while no such change is observed for non-oxyimino-cephalosporins. Mutational studies further confirm that this substrate-induced structural change is directly responsible for the positive fluorescence signal specific to oxyimino-cephalosporins. Our data provide mechanistic evidence to support the long-standing model that the evolutionary strategy of mutating the ⍀-loop leads to increased structural flexibility of this region, which in turn facilitates the binding of extended spectrum ␤-lactam antibiotics. The oxyiminocephalosporin-specific fluorescence profile of our engineered ␤-lactamase also demonstrates the possibility of designing substrate-selective biosensing systems.

Variations within Class-A β-Lactamase Physiochemical Properties Reflect Evolutionary and Environmental Patterns, but not Antibiotic Specificity

“…While conservation of function is the primary driving force in the evolution of most protein families, rampant antibiotic overuse has introduced new pressures leading to new resistance activities that reflect subtle differences within substrate specificity. The bulk of these changes are trivially explained by steric changes within the BL active site [52]; however, it has never been determined if antibiotic specificities are related to global physiochemical properties. We clearly demonstrate that they are not.…”

Deletion Mutations Conferring Substrate Spectrum Extension in the Class A β-Lactamase

“…3). We postulate that higher conformational flexibility in the mutant omega loop increased the accessibility of the binding pocket for the bulky C7␤ side chain of ceftazidime (31)(32)(33)(34).…”

Substrate Spectrum Extension of PenA in Burkholderia thailandensis with a Single Amino Acid Deletion, Glu168del

“…We postulate that the removal of the ␣-helix structure and increased space in the omega loop may accompany increased flexibility of the loop in the mutant enzymes. This flexibility would then, in turn, relieve steric hindrance between the omega loop and the bulky 7␤ side chain of ceftazidime, thereby increasing the accessibility of ceftazidime to the binding pocket (25).…”