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Reference: Zhang Y, et al. (2013) Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (??') of Saccharomyces cerevisiae Ribonucleotide Reductase: CONTRIBUTION TO COFACTOR FORMATION AND INTERSUBUNIT ASSOCIATION WITHIN THE ACTIVE HOLOENZYME. J Biol Chem 288(20):13951-9

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Abstract


The small subunit (?2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2(III)-Y(?)) that initiates nucleotide reduction in the large subunit (a2) via a long range radical transfer (RT) pathway in the holo-(a2)m(?2)n complex. The C-terminal tails of ?2 are predominantly responsible for interaction with a2, 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 ? 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 (??'), of which only ? contains a cofactor, to address both of these issues. We demonstrate that neither ?-Tyr(376) nor ?'-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-??' and an in vivo viability assay show that ?-Tyr(376) is essential for RT, whereas Tyr(323) in ?' is not. Although the C-terminal tail of ?' is dispensable for cofactor formation and RT, it is essential for interactions with ? and a to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the ? and ?' C-terminal tails relative to a within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the ? ?' interface.

Reference Type
Journal Article
Authors
Zhang Y, An X, Stubbe J, Huang M
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