Structural Basis for Superoxide Activation of <i>Flavobacterium johnsoniae</i> Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor

Rose, Hannah; Ghosh, Munmun; Maggiolo, Ailiena O.; Pollock, Christopher J.; Blaesi, Elizabeth J.; Hajj, Viviane; Wei, Yifeng; Rajakovich, Lauren J.; Chang, Wei‐chen; Han, Yilin; Hajj, Mariana; Krebs, Carsten; Silakov, Alexey; Pandelia, Maria-Eirini; Bollinger, J. Martin; Boal, Amie K.

doi:10.1021/acs.biochem.8b00247

Cited by 42 publications

(117 citation statements)

References 84 publications

(237 reference statements)

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“…Conversely, in our F. ignava NrdB samples presented here, the Mn 2 III/IV signal is clearly the dominant metal species. These observations underscore the catalytic relevance of the Mn 2 III/IV site, and support the notion that the NrdBi proteins represent a new subclass of class I RNRs, denoted subclass Id (6,8,9).…”

Section: Discussionsupporting

confidence: 74%

“…A similar high valent Mn dimer was recently found to be present in NrdB from L. blandensis (8). Later, Boal and co-workers also reported a similar multiline signal in Flavobacterium johnsoniae class I RNR (9). However, in both of these latter cases the multiline feature represented only a fraction of the total metal content.…”

Section: Discussionmentioning

confidence: 79%

“…Currently, class I RNRs have been subclassified based on radical cofactor type (subclasses Ia, Ib, Ic and Id) or evolutionary history (subclasses NrdA/B followed by a small letter plus subclass NrdE/F) (1,6). Metal content does not always follow phylogeny as two unrelated Mn 2 subclasses exist, where one subclass contains a tyrosyl radical in the vicinity of a Mn III /Mn III center (Ib, NrdE/F), and another recently identified subclass (Id, NrdAi/Bi) contains a mixed valent Mn IV /Mn III metal center that harbors the unpaired electron (7)(8)(9). Moreover, in eukaryotic RNRs and several evolutionarily unrelated bacterial class I subclasses, the NrdB component contains a stable tyrosyl radical in the vicinity of a diferric metal center (Ia).…”

Section: Introductionmentioning

confidence: 99%

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Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

Grinberg

Lundin

Sahlin

et al. 2018

Preprint

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Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radicalgenerating subunit (NrdB) that together catalyse reduction of the four ribonucleotides to their corresponding deoxyribonucleotides. Facklamia ignava NrdB is an unprecedented fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-cone. Grx, which in general is encoded elsewhere in the genome than is the RNR operon, is a known physiological reductant of RNRs. Here we show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and interestingly also via a monothiol mechanism, although less efficiently. A Grx that utilizes either or of these two reaction mechanisms has to our knowledge not been observed with a native substrate before. The ATP-cone, which is commonly found as an N-terminal domain of the catalytic subunit of RNRs, is an allosteric on/off switch that promotes dNDP reduction in presence of ATP and inhibits the enzyme activity in presence of dATP. Here we show that dATP bound to the ATP-cone of F. ignava NrdB promotes formation of tetramers that are unable to form enzymatically competent complexes with F. ignava NrdA. The ATP-cone binds two molecules of dATP, but only one molecule of the activating nucleotide ATP. F. ignava NrdB contains the recently identified radical factor Mn 2 III/IV . We show that NrdA from the firmicute F. ignava can form a catalytically competent RNR with the Mn 2 III/IV -containing NrdB from the flavobacterium Leeuwenhoekiella blandensis.

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 79%

Section: Introductionmentioning

confidence: 99%

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Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

Grinberg

Lundin

Sahlin

et al. 2018

Preprint

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“…In class Ia and Ib R2 proteins, the metal cofactor carries out a one-electron oxidation of a nearby tyrosine residue, and the stable active state of the cofactor is [10, 11, 13, 20, 21]. In class Ic, however, the radical equivalent is instead stored in form of the Mn IV /Fe III state of the cofactor [16, 22], while available data suggests that class Id forms a Mn IV /Mn III cofactor [14, 15]. In contrast, the Mn/Fe cofactor of R2lox proteins can catalyze two-electron oxidations.…”

Section: Introductionmentioning

confidence: 99%

Ether cross-link formation in the R2-like ligand-binding oxidase

et al. 2018

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R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of MnII and FeII in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine–valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.Electronic supplementary materialThe online version of this article (10.1007/s00775-018-1583-3) contains supplementary material, which is available to authorized users.

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“…Class Ic instead forms a radical-equivalent Mn IV /Fe III high-valent oxidation state of the metal site (15,16). A class Id, containing a Mn IV /Mn III cofactor, was also recently proposed (17)(18)(19). The dinuclear metal sites in classes Ia and Ic perform direct oxygen activation while class Ib requires an additional flavoprotein, NrdI, to generate superoxide used to oxidize the dimanganese site, ultimately resulting in tyrosyl radical generation (20)(21)(22).…”

Section: Main Textmentioning

confidence: 99%

Metal-independent ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

Srinivas

Lebrette

Lundin

et al. 2018

Preprint

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Ribonucleotide reductase (RNR) catalyzes the only known de-novo pathway for production of all four deoxyribonucleotides required for DNA synthesis. In aerobic RNRs, a di-nuclear metal site is viewed as an absolute requirement for generating and stabilizing an essential catalytic radical.Here we describe a new group of RNRs found in Mollicutes, including Mycoplasma pathogens, that possesses a metal-independent stable radical residing on a modified tyrosyl residue.Structural, biochemical and spectroscopic characterization reveal a stable DOPA radical species that directly supports ribonucleotide reduction in vitro and in vivo.The cofactor synthesis and radical generation processes are fundamentally different from established RNRs and require the flavoprotein NrdI. Several of the pathogens encoding this RNR variant are involved in diseases of the urinary tract and genitalia. Conceivably, this remarkable RNR variant provides an advantage under metal starvation induced by the immune system. We propose that the new RNR subclass is denoted class Ie.Here we describe a new group of RNR proteins in Mollicutes, including Mycoplasma pathogens, that possesses a metal-independent stable radical residing on a modified tyrosyl residue.Structural, biochemical and spectroscopic characterization reveal an unprecedented and remarkably stable DOPA radical species that directly supports ribonucleotide reduction in vitro and in vivo. We propose that the new RNR subclass is denoted class Ie. A new RNR subclass able to rescue an Escherichia coli strain lacking aerobic RNRSequence analysis revealed a group of class I RNR operons, present in common human pathogens e.g. Mycoplasma genitalium, Mycoplasma pneumoniae, and Streptococcus pyogenes.Analogous to standard class Ib RNRs, the operons contain the genes nrdE, nrdF and nrdI, coding for proteins R1, R2 and NrdI, respectively. Phylogenetically, the group forms a clade derived from class Ib proteins ( Fig S1). Strikingly, unlike any previously identified nrdE/F genes, the R2 proteins encoded by this group retain only 3 of the 6 metal binding residues that are otherwise completely conserved, and individually essential, regardless of R2 subclass. Two variants are observed in which 3 of the 4 carboxylate metal ligands are either substituted for valine, proline and lysine (VPK variant) or for glutamine, serine and lysine (QSK variant) ( Fig 1A). In both cases, the substitutions render the site formally charge neutral, a drastic change from the -4 net charge of the metal binding site in other R2 proteins. These substitutions appear to exclude a metal site and a radical generation mechanism even remotely similar to any thus far studied ribonucleotide reductase. Apart from the metal binding site, sequence and structural analysis of the R1, R2 and NrdI proteins show that the residues normally required for activity, including the R1 active site and the radical transfer path between the R1 and R2 subunits are conserved.As common for class I RNRs, several genomes encoding the QSK or VPK variant also h...

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Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor

Cited by 42 publications

References 84 publications

Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

Ether cross-link formation in the R2-like ligand-binding oxidase

Metal-independent ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

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