NDLI: Characterization of trimethoprim resistant <i>E. coli</i> dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates

Content Provider	Royal Society of Chemistry (RSC)
Author	Ellington, Andrew Anderson, Amy Thyer, Ross Cammarata, Michael Lombardo, Michael Brodbelt, Jennifer S. Wright, Dennis
Copyright Year	2017
Abstract	Pathogenic Escherichia coli, one of the primary causes of urinary tract infections, has shown significant resistance to the most popular antibiotic, trimethoprim (TMP), which inhibits dihydrofolate reductase (DHFR). The resistance is modulated by single point mutations of DHFR. The impact of two clinically relevant mutations, P21L and W30R, on the activity of DHFR was evaluated via measurement of Michaelis–Menten and inhibitory kinetics, and structural characterization was undertaken by native mass spectrometry with ultraviolet photodissociation (UVPD). Compared to WT-DHFR, both P21L and W30R mutants produced less stable complexes with TMP in the presence of co-factor NADPH as evidenced by the relative abundances of complexes observed in ESI mass spectra. Moreover, based on variations in the fragmentation patterns obtained by UVPD mass spectrometry of binary and ternary DHFR complexes, notable structural changes were localized to the substrate binding pocket for W30R and to the M20 loop region as well as the C-terminal portion containing the essential G–H functional loop for the P21L mutant. The results suggest that the mutations confer resistance through distinctive mechanisms. A novel propargyl-linked antifolate compound 1038 was shown to be a reasonably effective inhibitor of the P21L mutant.
Starting Page	4062
Ending Page	4072
Page Count	11
File Format	HTM / HTML PDF
ISSN	20416520
Volume Number	8
Issue Number	5
Journal	Chemical Science
DOI	10.1039/c6sc05235e
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Subscribed
Subject Keyword	Trimethoprim Enzyme inhibitor Michaelis Photodissociation Ultraviolet BMW M20 C-terminus Pathogenic Escherichia coli Antifolate Dihydrofolic acid Urinary tract infection Antibiotics Nicotinamide adenine dinucleotide phosphate TMP ESI Mutation Mass spectrometry
Content Type	Text
Resource Type	Article
Subject	Chemistry

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