Alpha-pyridine nucleotides as substrates for a plasmid-specified dihydrofolate reductase.

Smith, Sheila L.; Burchall, James J.

doi:10.1073/pnas.80.15.4619

Cited by 26 publications

(29 citation statements)

References 35 publications

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“…In addition to these electrostatic interactions, one of the adenosyl-2′-phosphate oxygen atoms is positioned 3.0 Å from the amide nitrogen of Ala36 (chain D). Specific interactions with this phosphate are required to explain the 20-fold preference of the enzyme for NADPH over NADH (30,32,33). The adenine base is positioned so that one side is solvent exposed, while the other side lies directly over Ala72-Ala73 (chain D), consistent with the large amide chemical shift previously observed in NMR studies of the R67 DHFR•NADP + complex (8).…”

Section: Substrate/cofactor Recognitionsupporting

confidence: 64%

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR•DHF•NADP + catalytic complex, resolved to1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67′-Y69′ residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationallychallenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation. KeywordsR67 DHFR; Dihydrofolate Reductase; X-ray crystallography; ternary complex; Type II DHFR Antifolate drug therapy plays a critical role in the treatment of pathogenic and neoplastic diseases. The evolution of a plasmid-encoded, Type II dihydrofolate reductase (DHFR) provides one mechanism for bacterial evasion of drugs such as trimethoprim that target the bacterial dihydrofolate reductase enzyme (1-4). Type II DHFR is an extremely unusual enzyme that exhibits no apparent structural or evolutionary relationship with the type I (chromosomal) enzyme. It is one of the smallest enzymes known to self-assemble into an active quaternary structure, forming a homotetramer consisting of four 78-residue peptides * To whom correspondence should be addressed. This type of active site structure also creates substantial evolutionary and mutational challenges to the enzyme. Since each mutation will alter four active site residues at a time, most of the evolutionary pressure that would normally optimize enzyme function is compromised by the need to balance the effects of substitutions at all four symmetry-related sites. For example, a residue substitution on one monomer, which might promote folate N5 protonation, may also interfere with NADPH binding when it is prese...

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Section: Substrate/cofactor Recognitionsupporting

confidence: 64%

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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“…Stacking between the nicotinamide ring of cofactor and the pteridine ring of folate is predicted in this model, consistent with ILOE constraints. [24] Binding promiscuity in R67 DHFR is also supported by the observation that it can utilize a-NADPH as cofactor [30] as well as be inhibited by novobiocin (K i = 70 mm) and congo red (K i = 2 mm). Neither of the last two ligands resembles NADPH or folate.…”

Section: R67 Dhfrmentioning

confidence: 71%

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Howell

2005

ChemBioChem

117

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There are numerous examples of proteins that catalyze the same reaction while possessing different structures. This review focuses on two dihydrofolate reductases (DHFRs) that have disparate structures and discusses how the catalytic strategies of these two DHFRs are driven by their respective scaffolds. The two proteins are E. coli chromosomal DHFR (Ec DHFR) and a type II R-plasmid-encoded DHFR, typified by R67 DHFR. The former has been described as a very well evolved enzyme with an efficiency of 0.15, while the latter has been suggested to be a model for a "primitive" enzyme that has not yet been optimized by evolution. This comparison underlines what is important to catalysis in these two enzymes and concurrently highlights fundamental issues in enzyme catalysis.

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“…ThioNADP(H) has been used as an inactive cofactor for NADPH in experiments using chicken, mouse, Escherichia coli, and Lactobacillus casei, and it has been shown to bind to the cofactor binding site (Dunn et al, 1978;Smith and Burchall, 1983;Thillet et al, 1990;McTigue et al, 1993). However, when we tested the effect of NADPS on DHFR enzyme activity, only slight inhibition of human DHFR was observed even with 180 mM NADS or NADPS (data not shown).…”

Section: The Effect Of Nad/nadp Analogs On Dhfr Activity and Expressionmentioning

confidence: 86%

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Hsieh

Tedeschi²,

Lawal³

et al. 2012

Mol Pharmacol

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Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin's lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.

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Alpha-pyridine nucleotides as substrates for a plasmid-specified dihydrofolate reductase.

Cited by 26 publications

References 35 publications

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

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