Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Howell, Elizabeth E.

doi:10.1002/cbic.200400237

Cited by 63 publications

(120 citation statements)

References 104 publications

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“…As we have shown, formation of the transition state is compromised upon addition of osmolytes. This sensitivity to water content suggests R67 DHFR is not very efficient in vivo, supporting our previous hypothesis that R67 DHFR is a primitive enzyme (4).…”

Section: Discussionsupporting

confidence: 86%

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A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH⅐DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.R67 dihydrofolate reductase, a type II DHFR, 2 catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. The gene for this enzyme is carried by an R-plasmid, and its presence confers resistance to the antibiotic drug, trimethoprim (TMP). Trimethoprim is a competitive inhibitor of chromosomal DHFR with a picomolar K i (1). Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2, 3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).R67 DHFR is a homotetramer with a single active site pore; the overall structure possesses 222 symmetry as seen in Fig. 1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH. This can be observed as R67 DHFR binds a total of two ligands as follows: either two NADPH molecules or two folate/DHF molecules or one NADPH plus one folate/DHF molecule (6). The first two complexes are dead-end (binary) complexes, whereas the third is the productive ternary complex. Because of the 222 symmetry, binding to either ligand is unlikely to be optimal.The active site pore of R67 DHFR is unusual in its hourglass shape as well as its large size (2938 Å 3 for apoR67 DHFR with hydrogens added, calculated by CASTp (4, 7)). Because of this large volume, DHF and NADPH cannot occupy all the space in the pore and must use water to mediate some contacts with the protein.How do substrate and cofactor bind to R67 DHFR? From NMR, crystallography, and docking studies, the pteridine ring of DHF/fo...

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Section: Discussionsupporting

confidence: 86%

“…Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2,3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).…”

mentioning

confidence: 99%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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“…Several of the known dfrA genes are found in gene cassettes (3). Members of the second, smaller group, DfrB (encoded by dfrB genes), are proteins of 78 aa that form a tetramer that binds both the substrate, dihydrofolate, and the cofactor, NADP, in equivalent positions, thus allowing reduction of the dihydrofolate to occur (1,5). The five known dfrB genes (Table 1), which are all found in gene cassettes, confer resistance to substantially lower levels of trimethoprim than the dfrA genes (1).…”

mentioning

confidence: 99%

New Integron-Associated Gene Cassette Encoding a Trimethoprim-Resistant DfrB-Type Dihydrofolate Reductase

Levings

Lightfoot

Elbourne

et al. 2006

Antimicrob Agents Chemother

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“…Trimethoprim inhibits bacterial dihydrofolate reductases. One mechanism of resistance is by exogenous expression of another DHFR that is not susceptible to the drug (12,13). Two types of genetically and structurally unrelated bacterial DHFR proteins have been characterized (12).…”

Section: Resultsmentioning

confidence: 99%

“…One band migrated at the estimated molecular mass of 25 kDa, while a second band migrated at twice this molecular mass, suggesting that this protein may form a dimer. Type I DHFRs are generally thought to exist as monomers within the cell, with only very few examples of dimerized forms of these enzymes (12,20,33).…”

Section: Resultsmentioning

confidence: 99%

Wide Variation in Antibiotic Resistance Proteins Identified by Functional Metagenomic Screening of a Soil DNA Library

McGarvey

Queitsch

Fields

2012

Appl Environ Microbiol

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cMost genes for antibiotic resistance present in soil microbes remain unexplored because most environmental microbes cannot be cultured. Only recently has the identification of these genes become feasible through the use of culture-independent methods. We screened a soil metagenomic DNA library in an Escherichia coli host for genes that can confer resistance to kanamycin, gentamicin, rifampin, trimethoprim, chloramphenicol, or tetracycline. The screen revealed 41 genes that encode novel protein variants of eight protein families, including aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, and transporters. Several proteins of the same protein family deviate considerably from each other yet confer comparable resistance. For example, five dihydrofolate reductases sharing at most 44% amino acid sequence identity in pairwise comparisons were equivalent in conferring trimethoprim resistance. We identified variants of aminoglycoside acetyltransferases and transporters that differ in the specificity of the drugs for which they confer resistance. We also found wide variation in protein structure. Two forms of rifampin ADP-ribosyltransferases, one twice the size of the other, were similarly effective at conferring rifampin resistance, although the short form was expressed at a much lower level. Functional metagenomic screening provides insight into the large variability in antibiotic resistance protein sequences, revealing divergent variants that preserve protein function.

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Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Cited by 63 publications

References 104 publications

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

New Integron-Associated Gene Cassette Encoding a Trimethoprim-Resistant DfrB-Type Dihydrofolate Reductase

Wide Variation in Antibiotic Resistance Proteins Identified by Functional Metagenomic Screening of a Soil DNA Library

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