Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.

Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major...

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Autores principales:	Christian Feiler, Adam C Fisher, Jason T Boock, Matthew J Marrichi, Lori Wright, Philipp A M Schmidpeter, Wulf Blankenfeldt, Martin Pavelka, Matthew P DeLisa
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1c03d57a53194acba870655dde9b2e96
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spelling	oai:doaj.org-article:1c03d57a53194acba870655dde9b2e962021-11-18T08:56:58ZDirected evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.1932-620310.1371/journal.pone.0073123https://doaj.org/article/1c03d57a53194acba870655dde9b2e962013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24023821/?tool=EBIhttps://doaj.org/toc/1932-6203Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A β-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to β-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F) that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a 'gatekeeper' residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.Christian FeilerAdam C FisherJason T BoockMatthew J MarrichiLori WrightPhilipp A M SchmidpeterWulf BlankenfeldtMartin PavelkaMatthew P DeLisaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 9, p e73123 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Christian Feiler Adam C Fisher Jason T Boock Matthew J Marrichi Lori Wright Philipp A M Schmidpeter Wulf Blankenfeldt Martin Pavelka Matthew P DeLisa Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.
description	Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A β-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to β-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F) that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a 'gatekeeper' residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.
format	article
author	Christian Feiler Adam C Fisher Jason T Boock Matthew J Marrichi Lori Wright Philipp A M Schmidpeter Wulf Blankenfeldt Martin Pavelka Matthew P DeLisa
author_facet	Christian Feiler Adam C Fisher Jason T Boock Matthew J Marrichi Lori Wright Philipp A M Schmidpeter Wulf Blankenfeldt Martin Pavelka Matthew P DeLisa
author_sort	Christian Feiler
title	Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.
title_short	Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.
title_full	Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.
title_fullStr	Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.
title_full_unstemmed	Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.
title_sort	directed evolution of mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/1c03d57a53194acba870655dde9b2e96
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Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.

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