Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical

Blaesi, Elizabeth J.; Palowitch, G.M.; Kai, Hu; Kim, Amelia J.; Rose, Hannah; Alapati, R.B.; Lougee, Marshall G.; Kim, Hee Jong; Taguchi, Alexander T.; Tan, Kong Ooi; Laremore, Tatiana N.; Griffin, Robert G.; Krebs, Carsten; Matthews, Megan L.; Silakov, Alexey; Bollinger, J. Martin; Allen, Benjamin D.; Boal, Amie K.

doi:10.1073/pnas.1811993115

Cited by 54 publications

(73 citation statements)

References 38 publications

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“…In the recently proposed class Id RNRs, which also bind a dimanganese cofactor, the radical equivalent appears to be stored at the metal site rather than at a tyrosine [ 18 , 19 ]. Notably, a metal-free class Ie was also recently discovered [ 20 – 22 ]. The third metal combination identified to date is a heterodinuclear manganese/iron cofactor with the manganese ion occupying the N-terminal metal-binding site (site 1) [ 23 – 26 ].…”

Section: Introductionmentioning

confidence: 99%

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine–valine ether cross-link formation in the R2-like ligand-binding oxidase

et al. 2019

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R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O 2 ) activation, catalyzes formation of a tyrosine–valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O 2 , but prefers manganese in the presence of O 2 , whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O 2 , while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O 2 . The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation. Electronic supplementary material The online version of this article (10.1007/s00775-019-01639-4) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine–valine ether cross-link formation in the R2-like ligand-binding oxidase

et al. 2019

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“…In subclass Ic (R2c), which contains a mixed manganese/iron complex (15)(16)(17), and probably also in the recently proposed dimanganese subclass Id (18,19), the metal complex itself acts as the catalytic radical, with the Mn ion shuttling between the III and IV oxidation states. A metal-free subclass Ie was also recently discovered (20)(21)(22).…”

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confidence: 96%

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

Kutin

Kositzki

Branca

et al. 2019

Journal of Biological Chemistry

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“…Two cysteines (on the bottom face of the RNR) provide the reducing equivalents to make dNDPs, and the third cysteine (on the top face of the RNR) is transiently oxidized to a thiyl radical (-S r ) that initiates NDP reduction (14). Distinct metallo-cofactors catalyze this oxidation (Figure 1c), and they are the primary basis for RNR classification (Ia-e, II, III), although a recently discovered nonmetallo-cofactor, 2,3-dihydroxy-phenylalanine radical (DOPA r ), breaks this paradigm (15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets

Greene

Kang

Cui

et al. 2020

Annu. Rev. Biochem.

135

195

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Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs’ central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.

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Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical

Cited by 54 publications

References 38 publications

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine–valine ether cross-link formation in the R2-like ligand-binding oxidase

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine–valine ether cross-link formation in the R2-like ligand-binding oxidase

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets

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