Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms and play an essential role in DNA replication and DNA repair. They are able to harness the amazing reactivity of free radicals to effect chemically difficult reactions with exquisite specificity. They utilize diverse metallo-cofactors to generate an essential protein-based thiyl radical that initiates substrate reduction. In class I RNRs, a diferric-tyrosyl radical cofactor is proposed to generate the thiyl radical by means of a 35 Å proton-coupled electron transfer pathway, which is unprecedented in biology. Because of their central role, they are also successful targets of several drugs used clinically in the treatment of a number of malignancies.

Current projects in the group include:

• Investigating the radical propagation pathway utilized by Class I RNRs through the use of unnatural amino acids using methodology developed by the Schultz lab (Scripps). (In collaboration with the Nocera lab.)
• Studying biosynthesis, activation, and regulation of Class I RNRs in E. coli and S. cerevisiae.
• Elucidating interactions between protein subunits of Class I RNR using photo-crosslinking.
• Examining regulation of RNRs: allosteric effetcs on subunit interactions, subunit localization, and tyrosyl radical concentration.
• Revealing mechanisms of clinically useful drugs: gemcitibine, hydroxyurea.

• Cotruvo, J.A.; Stubbe, J. An Active Dimanganese(III)-Tyrosyl Radical Cofactor in Escherichia coli Class Ib Ribonucleotide Reductase. Biochemistry Articles ASAP.
• Lohman, G.J.S.; Gerfen, G.J.; Stubbe, J. Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F₂CTP: adenosylcobalamin destruction and formation of a nucleotide based radical. Biochemistry Just Accepted.
• Lohman, G.J.S.; Stubbe, J. Inactivation of Lactobacillus leichmannii ribonucleotide reductase by F₂CTP: covalent modification. Biochemistry Just Accepted.
• Shanmugam, M.; Doan, P.E.; Lees, N.S.; Stubbe, J.; Hoffman, B.M. Identification of Protonated Oxygenic Ligands of Ribonucleotide Reductase Intermediate X. J. Am. Chem. Soc. 2009, 131, 3370-3376.
• Artin, E,; Wang, J.; Lohman, G.J.S.; Yokoyama, K.; Yu, G.; Griffin, R.G.; Bar, G.; Stubbe, J. Insight into the Mechanism of Inactivation of Ribonucleotide Reductase by Gemcitabine 5'-Diphosphate in the Presence or Absence of Reductant. Biochemistry 2009, 48, 11622-11629.
• Wang, J.; Lohman, G.J.S.; Stubbe, J. Mechanism of Inactivation of Human Ribonucleotide Reductase with p53R2 by Gemcitabine 5'-Diphosphate. Biochemistry 2009, 48, 11612-11621.
• Reece, S.Y.; Lutterman, D.A.; Seyedsayamdost, M.R.; Stubbe, J.; Nocera, D.G. Re(bpy)(CO)₃CN as a Probe of Conformational Flexibility in a Photochemical Ribonucleotide Reductase. Biochemistry 2009, 48, 5832-5838.
• Seyedsayamdost, M.R.; Stubbe, J. Replacement of Y730 and Y731 in the α2 subunit of Escherichia coli ribonucleotide reductase with 3-aminotyrosine using an evolved suppressor tRNA/tRNA-synthetase pair. Methods Enzymol. 2009, 462, 45-76.
• Seyedsayamdost, M.R.; Argirevic, T.; Minnihan, E.C.; Stubbe, J.; Bennati, M. Structural Examination of the Transient 3-Aminotyrosyl Radical on the PCET Pathway of E. coli Ribonucleotide Reductase by Multifrequency EPR Spectroscopy. J. Am. Chem. Soc. 2009, 131, 15729-15738.
• Zipse, H.; Artin, E.; Wnuk, S.; Lohman, G.J.S.; Martino, D.; Griffin, R.G.; Kacprzak, S.; Kaupp, M.; Hoffman, B.; Benatti, M.; Stubbe, J.; Lees, N. Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase. J. Am. Chem. Soc. 2009, 131, 200-211.
• Minnihan, E.C.; Seyedsayamdost, M.R.; Stubbe, J. Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase. Biochemistry 2009, 48, 12125-12132.
• Lee, Y.D.; Wang, J.; Stubbe, J; Elledge, S.J. Dif1 is a DNA-damage-regulated facilitator of nuclear import for ribonucleotide reductase. Molecular Cell 2008, 32, 70-80.
• Hassan, A.Q.; Stubbe, J. Mapping the subunit interface of ribonucleotide reductase (RNR) using photo cross-linking. Bioorganic & Medicinal Chemistry Letters 2008, 18, 5923-5925.
• Hassan, A.Q.; Wang, Y.; Plate, L.; Stubbe, J. Methodology to probe subunit interactions in ribonucleotide reductases. Biochemistry 2008, 47, 13046-13055.
• Cotruvo, J.A. Jr.; Stubbe, J. NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 2008, 105, 14383-14388.
• Hristova D.; Wu, C.-H.; Jiang, W.; Krebs, C.; Stubbe, J. Importance of the maintenance pathway in the regulation of the activity of Escherichia coli ribonucleotide reductase. Biochemistry 2008, 47, 3989-3999.
• Seyedsayamdost, M.R.; Xie, J.; Chan, C.T.; Schultz, P.G.; Stubbe, J. Site-specific insertion of 3-aminotyrosine into subunit α2 of E. coli ribonucleotide reductase: direct evidence for involvement of Y730 and Y731 in radical propagation. J. Am. Chem. Soc. 2007, 129, 15060-15071.
• Wang, J.; Lohman, G.J.S.; Stubbe, J. Enhanced subunit interactions with gemcitabine-5’-diphosphate inhibit ribonucleotide reductases. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14324-14329.
• Wu, C.-H.; Jiang, W.; Krebs, C.; Stubbe, J. YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in E. coli ribonucleotide reductase. Biochemistry 2007, 46, 11577-11588.
• Seyedsayamdost, M.R.; Yee, C.S.; Reece, S.Y.; Nocera, D.G.; Stubbe, J. pH rate profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli ribonucleotide reductase: Evidence that Y356 is a redox-active amino acid along the radical propagation pathway. J. Am. Chem. Soc., 2006, 128, 1562-1568.
• Ortigosa, A.D.; Hristova, D.; Perlstein, D.L.; Zhang, Z.; Huang, M.; Stubbe, J. Determination of the in vivo stoichiometry of tyrosyl radical per ββ' in Saccharomyces cerevisiae ribonucleotide reductase. Biochemistry 2006, 45, 12282-12294.
• Stubbe, J.; Nocera, D.G.; Yee, C.S.; Chang, M.C. Radical initiation in the class I ribonucleotide reductase: long-range proton-coupled electron transfer? Chem. Rev. 2003, 103, 2167-2201.

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Polyhydroxyalkanoates (PHAs), including polyhydroxybutyrates (PHBs) and polyhydroxyvalerates (PHVs), are biodegradable polymers with thermoplastic and/or elastomeric properties. They are synthesized by various bacteria under nutrient-limited conditions as a carbon storage material and are deposited as amorphous granules within the bacteria. Currently, companies such as Metabolix and Proctor and Gamble are pursuing the commercialization of PHAs as competitive plastics. Our research aim is to understand the mechanisms of PHA homeostasis. These mechanisms provide an important paradigm for non-template driven polymerization processes in biology. Ultimately, we would like to apply our knowledge about PHA homeostasis to engineer new biodegradable polymers and produce them in an economically competitive fashion.

Tools we use in the PHB project:

• Pre-steady state kinetics
• Biochemical assays
• Organic synthesis
• X-ray crystallography
• Transmission electron microscopy
• Fluorescence microscopy
• Molecular biology

• Li, P., Chakraborty, S., & Stubbe, J. Detection of Covalent and Noncovalent Intermediates in the Polymerization Reaction Catalyzed by a C149S Class III Polyhydroxybutyrate Synthase. Biochemistry 2009 48, 9202-9211.
• Tian, J., Sinskey, A. J., & Stubbe, J. Kinetic studies of polyhydroxybutyrate granule formation in Ralstonia eutropha H16 by transmission electron microscopy. J. Bacteriol. 2005 187, 3814-3824.
• Tian, J., He, A., Lawrence, A. G., Liu, P., Watson, N., Sinskey, A. J., & Stubbe, J. Analysis of transient polyhydroxybutyrate production in Ralstonia eutropha H16 by quantitative western analysis and transmission electron microscopy. J. Bacteriol. 2005, 187, 3825-3832.
• Stubbe, J., Tian, J., He, A., Sinskey, A. J., Lawrence, A. G., & Liu, P. Nontemplate-dependent polymerization processes: polyhydroxyalkanoate synthases as a paradigm. Annu. Rev. Biochem. 2005, 74, 433-480.
• Stubbe, J. & Tian, J. Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase. Nat. Prod. Rev. 2003, 20, 445-457.