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Structural basis for activation of class Ib ribonucleotide reductase.

ABSTRACT: The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn(III)2-tyrosyl radical (Y•) or a Fe(III)2-Y• cofactor in the NrdF subunit. Whereas Fe(III)2-Y• can self-assemble from Fe(II)2-NrdF and O2, activation of Mn(II)2-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O2. The crystal structures reported here of E. coli Mn(II)2-NrdF and Fe(II)2-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of Mn(II)2-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF Mn(II)2 active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of Mn(III)2-Y• cofactor assembly.

SUBMITTER: Boal AK

PROVIDER: S-EPMC3020666 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Structural basis for activation of class Ib ribonucleotide reductase.

Boal Amie K AK Cotruvo Joseph A JA Stubbe JoAnne J Rosenzweig Amy C AC

Science (New York, N.Y.) 20100805 5998

The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn(III)2-tyrosyl radical (Y•) or a Fe(III)2-Y• cofactor in the NrdF subunit. Whereas Fe(III)2-Y• can self-assemble from Fe(II)2-NrdF and O2, activation of Mn(II)2-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O2. The crystal structures reported here of E. coli Mn(II)2-NrdF and Fe(II)2-NrdF reveal differe ...[more]

PMID: 20688982

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Project description:Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (Fe(III)(2)-Y(•)) and dimanganese(III)-Y(•) (Mn(III)(2)-Y(•)) cofactors in the ?2 subunit, NrdF [Cotruvo, J. A., Jr., and Stubbe, J. (2010) Biochemistry 49, 1297-1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the Mn(III)(2)-Y(•) cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 ?g of NrdF was isolated with ?95% purity, with 0.2 Y(•)/?2, 0.9 Mn/?2, and a specific activity of 720 nmol min(-1) mg(-1). Under these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the Mn(III)(2)-Y(•) cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo.

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An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase.

Project description:Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is expressed under iron-limited and oxidative stress conditions. This RNR is composed of two homodimeric subunits: alpha2 (NrdE), where nucleotide reduction occurs, and beta2 (NrdF), which contains an unidentified metallocofactor that initiates nucleotide reduction. nrdE and nrdF are found in an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (His)(6)-NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with Mn(II) and incubated with fully reduced NrdI (NrdI(hq)) and O(2). Active RNR was rapidly produced with 0.25 +/- 0.03 tyrosyl radical (Y*) per beta2 and a specific activity of 600 units/mg. EPR and biochemical studies of the reconstituted cofactor suggest it is Mn(III)(2)-Y*, which we propose is generated by Mn(II)(2)-NrdF reacting with two equivalents of HO(2)(-), produced by reduction of O(2) by NrdF-bound NrdI(hq). In the absence of NrdI(hq), with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with Fe(II) and incubated with O(2) in the presence or absence of NrdI(hq) gave 0.2 and 0.7 Y*/beta2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdI(hq) hinders Fe(III)(2)-Y* cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the Mn(III)(2)-Y* cofactor, not the diferric-Y* one, is the active metallocofactor in vivo.

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Structural basis for assembly of the Mn(IV)/Fe(III) cofactor in the class Ic ribonucleotide reductase from Chlamydia trachomatis.

Project description:The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) employs a Mn(IV)/Fe(III) cofactor in each monomer of its β2 subunit to initiate nucleotide reduction. The cofactor forms by reaction of Mn(II)/Fe(II)-β2 with O2. Previously, in vitro cofactor assembly from apo β2 and divalent metal ions produced a mixture of two forms, with Mn at site 1 (Mn(IV)/Fe(III)) or site 2 (Fe(III)/Mn(IV)), of which the more active Mn(IV)/Fe(III) product predominates. Here we have addressed the basis for metal site selectivity by determining X-ray crystal structures of apo, Mn(II), and Mn(II)/Fe(II) complexes of Ct β2. A structure obtained anaerobically with equimolar Mn(II), Fe(II), and apoprotein reveals exclusive incorporation of Mn(II) at site 1 and Fe(II) at site 2, in contrast to the more modest site selectivity achieved previously. Site specificity is controlled thermodynamically by the apoprotein structure, as only minor adjustments of ligands occur upon metal binding. Additional structures imply that, by itself, Mn(II) binds in either site. Together, the structures are consistent with a model for in vitro cofactor assembly in which Fe(II) specificity for site 2 drives assembly of the appropriately configured heterobimetallic center, provided that Fe(II) is substoichiometric. This model suggests that use of a Mn(IV)/Fe(III) cofactor in vivo could be an adaptation to Fe(II) limitation. A 1.8 Å resolution model of the Mn(II)/Fe(II)-β2 complex reveals additional structural determinants for activation of the cofactor, including a proposed site for side-on (η(2)) addition of O2 to Fe(II) and a short (3.2 Å) Mn(II)-Fe(II) interionic distance, promoting formation of the Mn(IV)/Fe(IV) activation intermediate.

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