Streptococcus sanguinis Class Ib Ribonucleotide Reductase

Makhlynets, Olga V.; Boal, Amie K.; Rhodes, De Lacy V.; Kitten, Todd; Rosenzweig, Amy C.; Stubbe, Jo Anne

doi:10.1074/jbc.m113.533554

Cited by 41 publications

(31 citation statements)

References 67 publications

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“…6,7,9 More recently, in vitro assembly of a Mn III 2 -Y· cofactor using species specific NrdIs have been carried out in B. subtilis , 9,27 Streptococcus sanguinis , 53 B. anthracis , and B. cereus (24) with similar results. The interesting observation that a Fe III 2 -Y· cofactor can self-assemble in these enzymes in vitro and maintain catalytic activity, although demonstrably lower than the Mn-loaded cofactor (Supporting Information, SI-Table 1), 3−5,46,51 raises important biological questions as to the nature of the active cofactor in vivo and whether it changes with the growth conditions of the organism.…”

Section: Discussionmentioning

confidence: 70%

“…The results are distinct from all class Ib RNRs reported to date, which show no inhibition even at concentrations of 1 mM dATP. 5,49,50,53,60 Furthermore, with dATP and the B. anthracis RNR, the GEMMA analysis revealed individual subunits, αβ 2 , and α 2 β 2 , but no large quaternary states were reported. 24 Thus, the cause for the potent inhibition with the B. subtilis RNR requires further study.…”

Section: Discussionmentioning

confidence: 96%

“…25 We also note that we have observed a 20-fold difference between endogenous reductant and DTT with the S. sanguinis class Ib RNR. 53 …”

Section: Discussionmentioning

confidence: 99%

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Bacillus subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions

2014

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The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (MnIII2-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted MnIII2-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min–1 mg–1). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an “active” RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from MnIII2-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·/β2. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ∼1250 nmol min–1 mg–1 using a 1:1 mixture of NrdE:MnIII2-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (∼1 μM), NrdE is a monomer (α) and MnIII2-Y· NrdF is a dimer (β2). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations.

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Section: Discussionmentioning

confidence: 70%

Section: Discussionmentioning

confidence: 96%

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Bacillus subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions

2014

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“…The first such organism is Streptococcus sanguinis . 186 Although available intracellular Fe and Mn concentration data for this and other streptococci are not easily translatable to concentrations (μM) for comparison with the metal concentrations mentioned above, the related S. pneumoniae has been reported to accumulate similar levels of Mn and Fe (300 ng mg −1 protein), 187 suggesting the plausibility of metal loading and assembly of both Mn and Fe cofactors in vivo. Other studies of S. pneumoniae have reported somewhat higher Fe concentrations (900 ng mg −1 protein).…”

Section: Interplay Of Mn and Fe In Biological Systemsmentioning

confidence: 96%

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

2012

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How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use FeII as a Lewis acid under normal growth conditions but which switch to MnII under oxidative stress; (2) extradiol dioxygenases, which have been found to use both FeII and MnII, the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.

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“…NrdEF was shown to function in vitro with Fe 2+ , and if NrdI was included, with Mn 2+ (Makhlynets et al 2014). The Fe 3+ -cofactored form possessed 30% of the activity of the RNR with the Mn 3+ -cofactor.…”

Section: Biological Functions Of Mn2+ In Streptococcimentioning

confidence: 99%

Manganese uptake and streptococcal virulence

2015

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Streptococcal solute-binding proteins (SBPs) associated with ATP-binding cassette transporters gained widespread attention first as ostensible adhesins, next as virulence determinants, and finally as metal ion transporters. In this mini-review, we will examine our current understanding of the cellular roles of these proteins, their contribution to metal ion homeostasis, and their crucial involvement in mediating streptococcal virulence. There are now more than 35 studies that have collected structural, biochemical and/or physiological data on the functions of SBPs across a broad range of bacteria. This offers a wealth of data to clarify the formerly puzzling and contentious findings regarding the metal specificity amongst this group of essential bacterial transporters. In particular we will focus on recent findings related to biological roles for manganese in streptococci. These advances will inform efforts aimed at exploiting the importance of manganese and manganese acquisition for the design of new approaches to combat serious streptococcal diseases.

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Streptococcus sanguinis Class Ib Ribonucleotide Reductase

Cited by 41 publications

References 67 publications

Bacillus subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions

Bacillus subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

Manganese uptake and streptococcal virulence

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