Site-Specific Replacement of Y356 with 3,4-Dihydroxyphenylalanine in the β2 Subunit of E. coli Ribonucleotide Reductase

Seyedsayamdost, Mohammad R.; Stubbe, JoAnne

doi:10.1021/ja057776q

Cited by 88 publications

(171 citation statements)

References 16 publications

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“…RNR is inactivated by 0.5 eq of F 2 CDP/␣, as a consequence of altering the affinity of its two subunits and generating an asymmetric interaction within this complex. Recent studies using ␣ and ␤ substituted site specifically with unnatural amino acids have also been interpreted to suggest an asymmetry within the active RNR complex (19,20). Our results suggest that the paradigm of a 1:1 symmetrical complex between the two subunits of RNR must be re-evaluated.…”

Section: Discussionmentioning

confidence: 53%

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Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Wang

Lohman²,

Stubbe³

2007

Proc. Natl. Acad. Sci. U.S.A.

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175

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Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, ␣ and ␤, with proposed quaternary structures of ␣2␤2, ␣6␤2, or ␣6␤6, depending on the organism. The ␣ subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The ␤2 subunit houses the diferric Y • (1 radical per ␤2) cofactor that is required to initiate nucleotide reduction. 2 ,2 -Difluoro-2 -deoxycytidine (F2C) is presently used clinically in a variety of cancer treatments and the 5 -diphosphorylated F2C (F2CDP) is a potent inhibitor of RNRs. The studies with [1 -3 H]-F2CDP and [5-3 H]-F2CDP have established that F2CDP is a substoichiometric mechanism based inhibitor (0.5 eq F2CDP/␣) of both the Escherichia coli and the human RNRs in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 eq/␣) and is accompanied by release of 0.5 eq cytosine/␣. Inactivation also results in loss of 40% of ␤2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an ␣2␤2 tight complex is generated subsequent to enzyme inactivation by F2CDP, whereas in the human RNR, an ␣6␤6 tight complex is generated. Isolation of these complexes establishes that the weak interactions of the subunits in the absence of nucleotides are substantially increased in the presence of F2CDP and ATP. This information and the proposed asymmetry between the interactions of ␣n␤n provide an explanation for complete inactivation of RNR with substoichiometric amounts of F2CDP.G emcitabine, or 2Ј,2Ј-difluoro-2Ј-deoxycytidine (F 2 C), is a drug that is used clinically in the treatment of advanced pancreatic cancer and non-small cell lung carcinomas (1-3). In humans, F 2 C enters the cell via CNT-type or ENT-type transporters (4-6) and must be phosphorylated to exhibit its cytotoxicity. The monophosphate of F 2 C (F 2 CMP) is generated by deoxycytidine kinase (7) and is rapidly phosphorylated to the di-and triphosphates (F 2 CDP and F 2 CTP) (8, 9). Diphosphorylated F 2 C (F 2 CDP) is an irreversible inhibitor of ribonucleotide reductase (RNR) (10-13), and F 2 CTP functions as a chain terminator in the DNA polymerase reaction (14, 15). Differentially phosphorylated states of gemzar can also interfere with other enzymes involved in nucleotide metabolism. The mechanisms of cytotoxicity of F 2 C depend on the phosphorylated state of the inhibitor and are likely to be cell specific and multifactoral. Our recent synthesis of [1Ј-3 H]-F 2 CDP has provided the required tool to investigate the mechanism by which this molecule inactivates RNRs. Studies reported herein provide a previously unrecognized approach for RNR inhibition, one in which the mechanism based inhibitor (F 2 CDP) enhances the interactions between the two subunits of RNR preventing nucleotide reduction despite substoichiometric labeling.RNRs catalyze the conversion on nucleoside di(tri)phosphates to de...

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Section: Discussionmentioning

confidence: 53%

“…␤ is a homodimer that houses the diferric-tyrosyl radical (Y • ) required for initiating nucleotide reduction on ␣. Current evidence suggests that there is one Y • and 2 di-iron clusters/␤2 (19,20). The interactions between ␣ and ␤ are weak (K d of 0.2 M) in the absence of nucleotide (21).…”

mentioning

confidence: 99%

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Wang

Lohman²,

Stubbe³

2007

Proc. Natl. Acad. Sci. U.S.A.

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175

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“…The E. coli RNR is active as an α2β2 complex and nucleotide reduction is initiated by a long-range proton-coupled electron transfer (PCET) process in which Y 122 • in β2 reversibly oxidizes C 439 in α to a thiyl radical through a proposed pathway (Y 122 → W 48 → Y 356 within β to Y 731 → Y 730 → C 439 within α, Figure 1). 4,5 Our studies have shown that this process is masked by a rate-limiting conformational change (or changes) and that the conformational change is initiated by the binding of the substrate and allosteric effector to α 6-9. Conformational changes thus mask the PCET process.…”

Section: Introductionmentioning

confidence: 97%

A Hot Oxidant, 3-NO₂Y₁₂₂ Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

2010

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“…This peptide contains the 20 C-terminal residues of β2, including both the binding determinant for β2 to α2 and a key tyrosine residue at the N terminus of the peptide. This tyrosine residue is in a position analogous to Y356 in β2, which facilitates radical transport at the α2∶β2 interface (22). By appending a photooxidant (PO) to this peptide (PO-Y-βC19), the equivalent of •Y356 (•Y-βC19) can be photochemically generated.…”

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confidence: 99%

Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

Pizano

Lutterman

Holder

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemically within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has been labeled (98%) with a photooxidant ( Re tricarbonyl 1,10-phenanthroline methylpyridyl rhenium I ). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl) pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state 3 Re is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of protoncoupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates.proton-coupled electron transfer | radical generation | radical transport T he initiation and transport of many amino acid radicals occurs by proton-coupled electron transfer (PCET) (1-4). Accordingly, the importance of radicals in biological function (5) provides an imperative for the description of PCET in natural systems whose functions derive from radical-based chemistry (6). The PCET activity of amino acid radicals originates from the dependence of the reduction potential on the pK a of the amino acid in its oxidized and reduced states as well as the intrinsic redox potentials of the amino acid in its protonated and deprotonated states, and its association with hydrogen bonded partners as has been shown in proteins (7,8), β-hairpin peptides with interstrand dipolar contacts (9,10), and other de novo designed protein maquettes (11).Ribonucleotide reductases (RNRs) are essential enzymes of all organisms (12) that demonstrate exquisite control of radical transport for their function (13). RNRs catalyze the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs) and are therefore largely responsible for maintaining the cellular pool of monomeric DNA precursors. E. coli class Ia RNR consists of two homodimeric subunits, α2 and β2 (14,15). The α2 subunit contains the enzyme active site as well as two allosteric regulation sites, whereas β2 contains a diferric tyrosyl cofactor (•Y122), which is where the radical resides in the resting state of RNR. Nucleoside reduction requires formation of an α2∶β2 complex. Substrate turnover occurs by a radical mechanism mediated by an active site cysteine thiyl radical in α2 (•C439). A docking model (Fig. S1) based on crystal structure...

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Site-Specific Replacement of Y₃₅₆ with 3,4-Dihydroxyphenylalanine in the β2 Subunit of E. coli Ribonucleotide Reductase

Cited by 88 publications

References 16 publications

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

A Hot Oxidant, 3-NO₂Y₁₂₂ Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

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Site-Specific Replacement of Y356 with 3,4-Dihydroxyphenylalanine in the β2 Subunit of E. coli Ribonucleotide Reductase

Cited by 88 publications

References 16 publications

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

A Hot Oxidant, 3-NO2Y122 Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

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Site-Specific Replacement of Y₃₅₆ with 3,4-Dihydroxyphenylalanine in the β2 Subunit of E. coli Ribonucleotide Reductase

A Hot Oxidant, 3-NO₂Y₁₂₂ Radical, Unmasks Conformational Gating in Ribonucleotide Reductase