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Two β-lactamase variants with reduced clavulanic acid inhibition display different millisecond dynamics

DOI: 10.1128/AAC.02628-20 DOI Help

Authors: Wouter Elings (Leiden University) , Aleksandra Chikunova (Leiden University) , Danny B. Van Zanten (Leiden University) , Ralphe Drenth (Leiden University) , Misbha Ud Din Ahmad (Netherlands Cancer Institute) , Anneloes J. Blok (Leiden University) , Monika Timmer (Leiden University) , Anastassis Perrakis (Netherlands Cancer Institute) , Marcellus Ubbink (Leiden University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Antimicrobial Agents And Chemotherapy

State: Published (Approved)
Published: May 2021
Diamond Proposal Number(s): 19800

Open Access Open Access

Abstract: The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer most inhibitor resistance, K234R, as well as variant G132N that was found previously, were characterized using X-ray crystallography and NMR relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two, almost equally populated conformations that exchange with a rate of ca. 88 s−1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g. Ser104, Ser130) and a short loop (214-216). In the K234R mutant, the active site dynamics are significantly diminished with respect to the wild type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active site dynamics on the millisecond time scale are not required for catalytic function.

Journal Keywords: BlaC; NMR spectroscopy; inhibition; chemical exchange; directed evolution; error-prone PCR; crystallography

Diamond Keywords: Tuberculosis (TB); Bacteria; Enzymes

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 25/05/2021 14:03

Documents:
Antimicrobial Agents and Chemotherapy-2021-Elings-AAC.02628-20.full.pdf

Discipline Tags:

Catalysis Life Sciences & Biotech Health & Wellbeing Infectious Diseases Pathogens Structural biology Chemistry Biochemistry

Technical Tags:

Diffraction Macromolecular Crystallography (MX)