http://www.diva-portal.org This is the published version of a paper published in Journal of Biological Chemistry. Citation for the original published paper (version of record): Loderer, C., Jonna, V R., Crona, M., Grinberg, I R., Sahlin, M. et al. (2017) A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover Journal of Biological Chemistry, 292(46): 19044-19054 https://doi.org/10.1074/jbc.M117.806331 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-73795 ARTICLE cro Author’s Choice A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover Received for publication, July 12, 2017, and in revised form, September 28, 2017 Published, Papers in Press, October 2, 2017, DOI 10.1074/jbc.M117.806331 Christoph Loderer‡, Venkateswara Rao Jonna§, Mikael Crona‡1, Inna Rozman Grinberg‡, Margareta Sahlin‡, Anders Hofer§, Daniel Lundin‡2, and Britt-Marie Sjöberg‡3 From the ‡Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden and the §Department of Medical Biochemistry, Umeå University, SE-901 87 Umeå, Sweden Edited by Ruma Banerjee Ribonucleotide reductases (RNRs) catalyze the reduction of which are required for DNA synthesis and repair. Because this Downloaded from ribonucleotides to the corresponding deoxyribonucleotides, reaction is the only known biological pathway for de novo used in DNA synthesis and repair. Two different mechanisms deoxyribonucleotide synthesis, RNRs are considered to be help deliver the required electrons to the RNR active site. For- essential for every living organism. Today, three different mate can be used as reductant directly in the active site, or glu- classes of RNRs are known, which all share a common fold and taredoxins or thioredoxins reduce a C-terminal cysteine pair, a general reaction mechanism based on a thiyl radical (1). All http://www.jbc.org/ which then delivers the electrons to the active site. Here, we known RNRs, except a few viral RNRs, also share an allosteric characterized a novel cysteine-rich C-terminal domain (CRD), substrate-specificity regulation, where binding of a dNTP or which is present in most class II RNRs found in microbes. The ATP molecule modulates the specificity of the active site of the NrdJd-type RNR from the bacterium Stackebrandtia nassauen- enzyme (1). The three classes share a common ancestor (2); the sis was used as a model enzyme. We show that the CRD is main difference between them is their mechanism of radical involved in both higher oligomeric state formation and electron generation (1, 3). With the different radical, generating mech- at Stockholms Universitetsbibliotek on January 23, 2018 transfer to the active site. The CRD-dependent formation of anisms follow differences in dependence of oxygen and oligo- high oligomers, such as tetramers and hexamers, was induced by meric composition. addition of dATP or dGTP, but not of dTTP or dCTP. The elec- Class I RNRs (NrdAB/NrdEF) generate the radical in a sepa- tron transfer was mediated by an array of six cysteine residues at rate subunit (␤, NrdB/F) via a dimetal center that is oxidized by the very C-terminal end, which also coordinated a zinc atom. molecular oxygen. The radical is then transferred to the cata- The electron transfer can also occur between subunits, depend- lytic subunit (␣, NrdA/E) and back after the catalytic cycle. ␣ ␤ ing on the enzyme’s oligomeric state. An investigation of the Thus, this class is oxygen-dependent and forms active 2 2 native reductant of the system revealed no interaction with glu- heterotetramers. Terminal reduction of the oxidized active site taredoxins or thioredoxins, indicating that this class II RNR uses in ␣ is performed by thioredoxin or glutaredoxin systems at the a different electron source. Our results indicate that the CRD end of each turnover (4, 5). has a crucial role in catalytic turnover and a potentially new Class III RNRs (NrdD) also require a separate subunit (NrdG) terminal reduction mechanism and suggest that the CRD is to generate the radical. An iron–sulfur cluster in NrdG confers important for the activities of many class II RNRs. homolytic cleavage of the cofactor S-adenosyl-L-methionine (AdoMet) that generates a stable glycyl radical in the catalytic subunit. This glycyl radical is extremely sensitive toward oxy- Ribonucleotide reductases (RNRs)4 catalyze the reduction of gen, leading to protein backbone cleavage at oxygen exposition. ribonucleotides to the corresponding deoxyribonucleotides, ␣ NrdDs are described to be active as 2 homodimers and NrdG This work was supported by Swedish Research Council Grant 2016–01920 is only needed to generate the glycyl radical in NrdD (6, 7). and Swedish Cancer Society Grant CAN 2016/670 and by the Wenner-Gren Formate was shown to be a terminal reducing agent for some Foundations, Carl Trygger’s Foundation, and Kempe Foundations. The NrdD enzymes (8). Recently, class III RNRs that use a thiore- authors declare that they have no conflicts of interest with the contents of this article. doxin system for terminal reduction have been described (9). Author’s Choice—Final version free via Creative Commons CC-BY license. The RNR that is the focus of the current study, class II (NrdJ), This article contains supplemental Table S1 and supplemental data. generates the radical via homolytic cleavage of the vitamin B 1 Present address: Swedish Orphan Biovitrum AB, SE-112 76 Solna, Sweden. 12 2 To whom correspondence may be addressed. Tel.: 46-708-123-922; Fax: coenzyme (AdoCbl) within the enzyme, close to the active site. 46-8-155597; E-mail: [email protected]. After the catalytic cycle, AdoCbl is reconstituted (10). This 3 To whom correspondence may be addressed. Tel.: 46-8-164150; Fax: 46-8- mechanism makes NrdJ enzymes independent of oxygen, but 155597; E-mail: [email protected]. 4 The abbreviations used are: RNR, ribonucleotide reductase; AdoCbl, adeno- sylcobalamin; AdoMet, S-adenosyl-L-methionine; CRD, cysteine-rich C-ter- minal domain; GEMMA, gas phase electrophoretic macromolecule analy- class II RNR; NrdJd⌬CRD, the C-terminally deleted version of the S. sis; HED, 2-hydroxyethyl disulfide; ICP, inductively coupled plasma nassauensis class II RNR; SEC, size exclusion chromatography; TCEP, analysis; ICP-OES, inductively coupled plasma optical emission spectrom- tris(2-carboxyethyl)phosphine hydrochloride; IPTG, isopropyl 1-thio-␤-D- etry; NrdJd-wt, the full-length version of the Stackebrandtia nassauensis galactopyranoside. 19044 J. Biol. Chem. (2017) 292(46) 19044–19054 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Catalytic turnover in class II ribonucleotide reductase Downloaded from http://www.jbc.org/ at Stockholms Universitetsbibliotek on January 23, 2018 Figure 1. Abundance and sequence of CRD were studied by phylogenetic and sequence analysis of NrdJ and NrdJd. A, maximum likelihood phyloge- netic tree of a representative selection of NrdJ sequences. Subclasses with characterized members (NrdJm (green), NrdJd (red), and NrdJaϩb(yellow)) and candidate subclasses (cyan, purple, orange, and light blue) are indicated with color. Lactobacillus leichmannii RNR (Ll) is located in subclass NrdJm, whereas the RNR from Thermotoga maritima (Tm) is not part of a defined subclass. B, appearance of the cysteine-rich domain mapped on a phylogenetic tree of a selection of nonmonomeric NrdJ sequences. The selected subset is marked by the dotted line in A. Subclasses with characterized members (NrdJd (red) and NrdJaϩb (yellow)) and candidate subclasses (cyan, purple, orange, and light blue) are indicated with color. The presence of a CRD is indicated with a blue circle, absence with a white circle. The NrdJaϩb subclass has the encoded CRD as a separate gene. C, logo of the CRD derived from 217 nonredundant sequences from subclasses NrdJd and unclassified NrdJ. instead dependent on B12. The oligomeric organization of the class II RNRs contain a so far undescribed cysteine-rich C-ter- NrdJ enzymes is more diverse than in the other classes. Some minal domain (CRD). We show that the CRD is involved in representatives, such as the enzyme from Thermoplasma aci- oligomerization and the terminal reduction of the class II RNR ␣ dophilum, have been shown to be active as 2 homodimers (11); from Stackebrandtia nassauensis and that the CRD hence plays the NrdJ from Lactobacillus leichmannii is active as monomer, a crucial role in the catalytic cycle of class II RNRs. where an additional domain mimics the dimer interface, required for allosteric regulation (12). Higher oligomers have Results been observed for the full-length NrdJ from Thermotoga mari- Phylogenetic analysis tima, whereas a truncated variant utilized for X-ray crystallog- We estimated a maximum likelihood phylogenetic tree from raphy crystallized as a dimer (13). The terminal reductant is an alignment of sequences representing the full diversity of poorly studied for NrdJ enzymes. The monomeric NrdJ from NrdJ sequences at the 75% identity level (Fig. 1A; supplemental L. leichmannii was shown to receive electrons from thioredoxin data). The resolution increased after exclusion of sequences (14), but for other NrdJ enzymes reduction by thioredoxin or from the monomeric class II