2014
DOI: 10.1002/pro.2602
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Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active‐site substitutions

Abstract: Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct conse… Show more

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Cited by 10 publications
(13 citation statements)
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“…To access highly purified DfrB variants, we capitalized on the remarkable thermostability of DfrB1 and DfrB2, vividly illustrated by maintenance of activity upon boiling for 20 min. We purified the DfrBs to ≥95% using heat-precipitation of crude lysates at 65 to 75 °C (Table S4; Figure S1). This one-day purification method procured 6.2 to 12.4 mg (3.7 to 8.5 U) of all DfrBs from 200 mL-scale expressions in E. coli (Table S4, Table S5).…”
Section: Results and Discussionmentioning
confidence: 96%
“…To access highly purified DfrB variants, we capitalized on the remarkable thermostability of DfrB1 and DfrB2, vividly illustrated by maintenance of activity upon boiling for 20 min. We purified the DfrBs to ≥95% using heat-precipitation of crude lysates at 65 to 75 °C (Table S4; Figure S1). This one-day purification method procured 6.2 to 12.4 mg (3.7 to 8.5 U) of all DfrBs from 200 mL-scale expressions in E. coli (Table S4, Table S5).…”
Section: Results and Discussionmentioning
confidence: 96%
“…The most common resistance results from mutations leading to the synthesis of an altered form of DHFR with reduced affinity for an inhibitor or inhibitors [9]. Acquired resistance is also associated with mobile elements temporarily located on the chromosome by transposon movement, referred to as plasmid-encoded DHFR [1014]. …”
Section: Introductionmentioning
confidence: 99%
“…Phylogenetic analyses of DHFR [2023] as well as studies and simulations of evolutionary processes [14, 2430] have been performed to study the rapid development of resistance to antifolate agents in response to the excessive use of antibiotics [31]. Our efforts focus on predictive models of the evolution of drug resistance in bacterial pathogens having the goal of developing novel therapeutics targeting drug resistant strains in anticipation of their development in the wild [26, 27, 32].…”
Section: Introductionmentioning
confidence: 99%
“…These various experiments and constructs indicate the N-termini can be fused without a loss of function. Pelletier and co-workers have made similar dimeric fused constructs of R67 DHFR. , …”
mentioning
confidence: 99%