title: Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.
abstract: 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.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, United States of America.
authors.forename: Christian
authors.initials: C
authors.lastname: Feiler
authors.forename: Adam C
authors.initials: AC
authors.lastname: Fisher
authors.forename: Jason T
authors.initials: JT
authors.lastname: Boock
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Marrichi
authors.forename: Lori
authors.initials: L
authors.lastname: Wright
authors.forename: Philipp A M
authors.initials: PA
authors.lastname: Schmidpeter
authors.forename: Wulf
authors.initials: W
authors.lastname: Blankenfeldt
authors.forename: Martin
authors.initials: M
authors.lastname: Pavelka
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D055162:Biocatalysis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D019818:Clavulanic Acid / Q000494:pharmacology; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects; D009169:Mycobacterium tuberculosis / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D019818:Clavulanic Acid; D001618:beta-Lactamases
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14603-8
references.pmid: 18784364
references.citation: Protein Sci. 2006 Mar;15(3):449-58
references.pmid: 16452624
references.citation: J Biol Chem. 2007 Mar 16;282(11):8309-16
references.pmid: 17218314
references.citation: J Bacteriol. 2005 Mar;187(6):1892-900
references.pmid: 15743935
references.citation: Biochemistry. 2007 Oct 30;46(43):11998-2004
references.pmid: 17915954
references.citation: Antimicrob Agents Chemother. 1995 Dec;39(12):2620-4
references.pmid: 8592990
references.citation: Nat Struct Biol. 2001 Mar;8(3):238-42
references.pmid: 11224569
references.citation: Proc Natl Acad Sci U S A. 2003 May 13;100(10):6115-20
references.pmid: 12721369
references.citation: Protein Sci. 2010 May;19(5):1079-90
references.pmid: 20440843
references.citation: Cell. 2007 Mar 23;128(6):1037-50
references.pmid: 17382878
references.citation: J Antimicrob Chemother. 2000 Jan;45(1):1-4
references.pmid: 10629005
references.citation: PLoS One. 2008 Jun 11;3(6):e2351
references.pmid: 18545653
references.citation: Antimicrob Agents Chemother. 2006 Aug;50(8):2762-71
references.pmid: 16870770
references.citation: J Biol Chem. 1998 Jul 17;273(29):18003-6
references.pmid: 9660752
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Jan;66(Pt 1):22-5
references.pmid: 20057045
references.citation: Microbiology (Reading). 2005 Feb;151(Pt 2):521-532
references.pmid: 15699201
references.citation: Adv Protein Chem. 1970;24:1-95
references.pmid: 4912353
references.citation: Nature. 1994 Aug 4;370(6488):389-91
references.pmid: 8047147
references.citation: Nature. 1991 Jun 6;351(6326):456-60
references.pmid: 1904554
references.citation: Proc Natl Acad Sci U S A. 2012 Aug 14;109(33):13392-7
references.pmid: 22847444
references.citation: Science. 1994 Apr 15;264(5157):375-82
references.pmid: 8153624
references.citation: J Biol Chem. 1984 Feb 25;259(4):2149-54
references.pmid: 6365904
references.citation: Acta Crystallogr D Biol Crystallogr. 2003 Jul;59(Pt 7):1131-7
references.pmid: 12832755
references.citation: J Pathol Bacteriol. 1946 Jul;58(3):495-500
references.pmid: 20283086
references.citation: J Bacteriol. 2008 Oct;190(19):6428-38
references.pmid: 18658266
references.citation: J Biol Chem. 2007 Mar 16;282(11):7903-11
references.pmid: 17229735
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: EMBO J. 2004 Oct 13;23(20):3962-72
references.pmid: 15385959
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
references.pmid: 20124692
references.citation: Nat Struct Mol Biol. 2008 Oct;15(10):1109-18
references.pmid: 18794843
references.citation: J Bacteriol. 2006 Sep;188(18):6669-79
references.pmid: 16952959
references.citation: Biochem J. 1991 May 15;276 ( Pt 1):269-70
references.pmid: 2039479
references.citation: Nucleic Acids Res. 2009 Jul;37(Web Server issue):W441-5
references.pmid: 19395593
references.citation: J Bacteriol. 2005 Nov;187(22):7667-79
references.pmid: 16267291
references.citation: Antimicrob Agents Chemother. 2009 Jan;53(1):242-8
references.pmid: 18981261
references.citation: Science. 2009 Feb 27;323(5918):1215-8
references.pmid: 19251630
references.citation: Microbiology (Reading). 2007 Oct;153(Pt 10):3350-3359
references.pmid: 17906134
references.citation: Biochemistry. 1988 Oct 18;27(21):8063-8
references.pmid: 3233195
references.citation: J Biol Chem. 2008 Dec 12;283(50):35223-35
references.pmid: 18819916
references.citation: Genetics. 2002 Mar;160(3):823-32
references.pmid: 11901104