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Instruments / Technical Area	Publication Details	Published
	Structure of a ribonucleotide reductase R2 protein radical Hugo Lebrette , Vivek Srinivas , Juliane John , Oskar Aurelius , Rohit Kumar , Daniel Lundin , Aaron S. Brewster , Asmit Bhowmick , Abhishek Sirohiwal , In-Sik Kim , Sheraz Gul , Cindy Pham , Kyle D. Sutherlin , Philipp Simon , Agata Butryn , Pierre Aller , Allen M. Orville , Franklin D. Fuller , Roberto Alonso-Mori , Alexander Batyuk , Nicholas K. Sauter , Vittal K. Yachandra , Junko Yano , Ville R. I. Kaila , Britt-Marie Sjöberg , Jan Kern , Katarina Roos , Martin Högbom Science 382, 109 - 113 DOI: 10.1126/science.adh8160 Show Abstract ▼ Abstract: Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O–O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.	Oct 2023
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structural and biochemical investigation of class I ribonucleotide reductase from the hyperthermophile Aquifex aeolicus Daniel Rehling , Emma Rose Scaletti , Inna Rozman Grinberg , Daniel Lundin , Margareta Sahlin , Anders Hofer , Britt-Marie Sjöberg , Pal Stenmark Biochemistry DOI: 10.1021/acs.biochem.1c00503 Open Access Show Abstract ▼ Abstract: Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found in nearly all living organisms. Class I RNRs are composed of two proteins, a large α-subunit (R1) and a smaller β-subunit (R2) that exist as homodimers, that combine to form an active heterotetramer. Aquifex aeolicus is a hyperthermophilic bacterium with an unusual RNR encoding a 346-residue intein in the DNA sequence encoding its R2 subunit. We present the first structures of the A. aeolicus R1 and R2 (AaR1 and AaR2, respectively) proteins as well as the biophysical and biochemical characterization of active and inactive A. aeolicus RNR. While the active oligomeric state and activity regulation of A. aeolicus RNR are similar to those of other characterized RNRs, the X-ray crystal structures also reveal distinct features and adaptations. Specifically, AaR1 contains a β-hairpin hook structure at the dimer interface, which has an interesting π-stacking interaction absent in other members of the NrdAh subclass, and its ATP cone houses two ATP molecules. We determined structures of two AaR2 proteins: one purified from a construct lacking the intein (AaR2) and a second purified from a construct including the intein sequence (AaR2_genomic). These structures in the context of metal content analysis and activity data indicate that AaR2_genomic displays much higher iron occupancy and activity compared to AaR2, suggesting that the intein is important for facilitating complete iron incorporation, particularly in the Fe2 site of the mature R2 protein, which may be important for the survival of A. aeolicus in low-oxygen environments.	Dec 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site Markel Martinez-Carranza , Venkateswara Rao Jonna , Daniel Lundin , Margareta Sahlin , Lars-Anders Carlson , Newal Jemal , Martin Högbom , Britt-Marie Sjöberg , Pal Stenmark , Anders Hofer Journal Of Biological Chemistry DOI: 10.1074/jbc.RA120.014895 Diamond Proposal Number(s): [11265] Open Access Show Abstract ▼ Abstract: Ribonucleotide reductase (RNR) is a central enzyme for DNA building block synthesis. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, electron microscopy, x-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral, but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.	Sep 2020
B21-High Throughput SAXS I24-Microfocus Macromolecular Crystallography	Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens Vivek Srinivas , Hugo Lebrette , Daniel Lundin , Yuri Kutin , Margareta Sahlin , Michael Lerche , Jürgen Eirich , Rui M. M. Branca , Nicholas Cox , Britt-Marie Sjöberg , Martin Högbom Nature 47 DOI: 10.1038/s41586-018-0653-6 Diamond Proposal Number(s): [11265, 15806] Show Abstract ▼ Abstract: Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5,6,7. Here we describe a group of RNR proteins in Mollicutes—including Mycoplasma pathogens—that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR—some of which are developing resistance to antibiotics—are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.	Oct 2018

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