PortEco logo


From EcoliWiki
Jump to: navigation, search



Cotruvo, JA Jr and Stubbe, J (2010) An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase. Biochemistry 49:1297-309


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.


PubMed PMC3190568 Online version:10.1021/bi902106n


Catalytic Domain; Coenzymes/biosynthesis; Coenzymes/chemistry; Coenzymes/classification; Escherichia coli Proteins/biosynthesis; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/classification; Free Radicals/chemistry; Manganese Compounds/chemistry; Metalloproteins/biosynthesis; Metalloproteins/chemistry; Metalloproteins/classification; Multiprotein Complexes/chemistry; Multiprotein Complexes/classification; Oxidants/chemistry; Oxidation-Reduction; Oxygen/chemistry; Peroxides/chemistry; Protein Subunits/chemistry; Protein Subunits/classification; Ribonucleotide Reductases/biosynthesis; Ribonucleotide Reductases/chemistry; Ribonucleotide Reductases/classification; Tyrosine/chemistry


You can help EcoliWiki by summarizing why this paper is useful
This paper describes the first successful in vitro assembly of an active dimanganese(III)-tyrosyl radical cofactor in a class Ib ribonucleotide reductase, NrdEF. NrdF can also form a diferric-tyrosyl radical cofactor that is active in nucleotide reduction. While it is not yet clear whether NrdF contains iron or manganese (or both, depending on the growth conditions) in vivo, the paper demonstrates that NrdI is required for assembly of the manganese cofactor. By contrast, the diferric-tyrosyl radical cofactor does not require NrdI for its formation. The requirement for NrdI for dimanganese(III)-tyrosyl radical cofactor assembly provides a rationale for the universal conservation of nrdI in class Ib RNR systems and suggests that this role for NrdI may be relevant inside the cell.

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.


Gene product Qualifier GO ID GO term name Evidence Code with/from Aspect Notes Status

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

See also


See Help:References for how to manage references in EcoliWiki.