title: 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.
abstract: 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 other β-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 Ω-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 β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism.
authors.affiliation: From the Verna and Marrs McLean Department of Biochemistry and Molecular Biology and the Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 and.
authors.forename: Vlatko
authors.initials: V
authors.lastname: Stojanoski
authors.affiliation: the Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 and.
authors.forename: Dar-Chone
authors.initials: DC
authors.lastname: Chow
authors.affiliation: From the Verna and Marrs McLean Department of Biochemistry and Molecular Biology and.
authors.forename: Liya
authors.initials: L
authors.lastname: Hu
authors.affiliation: the Berkeley Center for Structural Biology, Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
authors.forename: Banumathi
authors.initials: B
authors.lastname: Sankaran
authors.affiliation: From the Verna and Marrs McLean Department of Biochemistry and Molecular Biology and.
authors.forename: Hiram F
authors.initials: HF
authors.lastname: Gilbert
authors.affiliation: From the Verna and Marrs McLean Department of Biochemistry and Molecular Biology and.
authors.forename: B V Venkataram
authors.initials: BV
authors.lastname: Prasad
authors.affiliation: From the Verna and Marrs McLean Department of Biochemistry and Molecular Biology and the Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 and timothyp@bcm.edu.
authors.forename: Timothy
authors.initials: T
authors.lastname: Palzkill
mesh_terms: D055162:Biocatalysis; D002442:Ceftazidime / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010406:Penicillins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D010406:Penicillins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D002442:Ceftazidime; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1
keywords: Antibiotic Resistance; Beta-Lactamase; Enzyme Catalysis; Enzyme Evolution; Enzyme Kinetics; Enzyme Structure; Protein Stability; Protein Structure; X-ray Crystallography