Dataset Information

A triple mutant in the ?-loop of TEM-1 ?-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis.

ABSTRACT: ?-Lactamases are bacterial enzymes that hydrolyze ?-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded ?-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other ?-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site ?-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine ?-lactamases. The results reveal that the robustness of the overall ?-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism.

SUBMITTER: Stojanoski V

PROVIDER: S-EPMC4400348 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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A triple mutant in the Ω-loop of TEM-1 β-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis.

Stojanoski Vlatko V Chow Dar-Chone DC Hu Liya L Sankaran Banumathi B Gilbert Hiram F HF Prasad B V Venkataram BV Palzkill Timothy T

The Journal of biological chemistry 20150220 16

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all ot ...[more]

PMID: 25713062

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Project description:Many studies have shown that the dynamic motions of individual protein segments can play an important role in enzyme function. Recent structural studies of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) demonstrate that PEPCK contains a 10-residue Omega-loop domain that acts as an active site lid. On the basis of these structural studies, we have previously proposed a model for the mechanism of PEPCK catalysis in which the conformation of this mobile lid domain is energetically coupled to ligand binding, resulting in the closed conformation of the lid, necessary for correct substrate positioning, becoming more energetically favorable as ligands associate with the enzyme. Here we test this model by introducing a point mutation (A467G) into the center of the Omega-loop lid that is designed to increase the entropic penalty for lid closure. Structural and kinetic characterization of this mutant enzyme demonstrates that the mutation has decreased the favorability of the enzyme adapting the closed lid conformation. As a consequence of this shift in the equilibrium defining the conformation of the active site lid, the enzyme's ability to stabilize the reaction intermediate is weakened, resulting in catalytic defect. This stabilization is initially surprising, as the lid domain makes no direct contacts with the enolate intermediate formed during the reaction. Furthermore, during the conversion of OAA to PEP, the destabilization of the lid-closed conformation results in the reaction becoming decoupled as the enolate intermediate is protonated rather than phosphorylated, resulting in the formation of pyruvate. Taken together, the structural and kinetic characterization of A467G-PEPCK supports our model of the role of the active site lid in catalytic function and demonstrates that the shift in the lowest-energy conformation between open and closed lid states is a function of the free energy available to the enzyme through ligand binding and the entropic penalty for ordering of the 10-residue Omega-loop lid domain.

Dataset Information

A triple mutant in the ?-loop of TEM-1 ?-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis.

Publications

A triple mutant in the Ω-loop of TEM-1 β-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis.

Similar Datasets

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