Two β-Lactamase Variants with Reduced Clavulanic Acid Inhibition Display Different Millisecond Dynamics

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 the mos...

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Bibliographic Details
Published in:	Antimicrobial agents and chemotherapy Vol. 65; no. 8; p. e0262820
Main Authors:	Elings, Wouter, Chikunova, Aleksandra, van Zanten, Danny B, Drenth, Ralphe, Ahmad, Misbha Ud Din, Blok, Anneloes J, Timmer, Monika, Perrakis, Anastassis, Ubbink, Marcellus
Format:	Journal Article
Language:	English
Published:	United States American Society for Microbiology 16-07-2021
Subjects:	beta-Lactamase Inhibitors - pharmacology beta-Lactamases - genetics Clavulanic Acid - pharmacology Crystallography, X-Ray Escherichia coli - genetics Mechanisms of Resistance Mycobacterium tuberculosis - genetics X-ray crystallography BlaC inhibition NMR spectroscopy error-prone PCR chemical exchange directed evolution
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Summary:	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 the most inhibitor resistance, K234R, as well as variant G132N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance (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 . 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 -conformation. The crystal structure suggests multiple conformations for several side chains (e.g., Ser104 and Ser130) and a short loop (positions 214 to 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Citation Elings W, Chikunova A, van Zanten DB, Drenth R, Ahmad MUD, Blok AJ, Timmer M, Perrakis A, Ubbink M. 2021. Two β-lactamase variants with reduced clavulanic acid inhibition display different millisecond dynamics. Antimicrob Agents Chemother 65:e02628-20. https://doi.org/10.1128/AAC.02628-20.
ISSN:	0066-4804 1098-6596
DOI:	10.1128/AAC.02628-20