title: Investigation of in vivo roles of the C-terminal tails of the small subunit (ββ') of Saccharomyces cerevisiae ribonucleotide reductase: contribution to cofactor formation and intersubunit association within the active holoenzyme.
abstract: The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2(III)-Y(•)) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr(356) in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ'), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr(376) nor β'-Tyr(323) (Tyr(356) equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ' and an in vivo viability assay show that β-Tyr(376) is essential for RT, whereas Tyr(323) in β' is not. Although the C-terminal tail of β' is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β' C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β' interface.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors.forename: Xiuxiang
authors.initials: X
authors.lastname: An
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045. Electronic address: mingxia.huang@ucdenver.edu.
authors.forename: Mingxia
authors.initials: M
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D005609:Free Radicals; D007501:Iron / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry
chemical_list: D005609:Free Radicals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases
keywords: Diferric Tyrosyl Cofactor; Iron; Long Range Radical Transfer; Nucleoside Nucleotide Biosynthesis; Radicals; Ribonucleotide Reductase; Tyrosine