General Research Interests:

• Mechanism of natural product DNA Cleavers used clinically: our interests include: 2D NMR methods to determine the structures of the drugs bound to DNA, synthesis and structure of the deoxyribose lesions generated by the drugs and mechanism of repair of these lesions
• Mechanism and Regulation of Ribonucleotide Ruductases: our interests include study of clinically active compounds that inactivate reductases; Mechanism of metallo-cofactor assembly in vivo; mechanism of radical initiation using technology to insert unnatural amino acids into the proteins; signal transduction cascades induced by DNA damaging agents;
• Polyester Biosynthesis: understanding non-template driven polymerization reactions and use of bioengineering methods to generate new biodegradable polymers;
• Mechanism, Structure, and Regulation of the Purine Biosynthetic Pathway: studies to understand the importance of transient protein-protein interactions in vivo.

Ribonucleotide Reductases (RNRs):
RNRs are enzymes that catalyze an essensial step in DNA replication and repair. They use unusual metallo-cofactors (a diferric tyrosyl radical, adenosylcobalamin and a glycyl radical). These cofactors serve as radical initiators to generate a cysteinyl radical that initiates radical dependent nucleotide reduction at homologous active sites. Studies are ongoing to understand: the biosynthetic pathway for diferric-tyrosyl radical cofactor assembly in vivo in yeast; the mechanism of radical initiation over 35 Å using unnatural amino acids and the regulation of dNTP production at all levels. Collaborative efforts with the Griffin, Nocera and Drennan labs are ongoing.

Mechanism of Action Anittumor Natural Products:
Metallo-bleomycins (BLMs), are natural products used clinically and act catalytically to destroy DNA. Physical organic methods have been used to understand the chemistry of DNA cleavage. Multidimensional NMR methods using colbalt-BLM and oligonucleotides have been used to elucidate the basis for the chemical and sequence specificity of DNA cleavage. These studies have resulted in a proposal for how one BLM can catalyze ds-DNA cleavage without dissociation from the DNA. NMR methods are being used to investigate the structure of the lesions produced in the DNA backbone by BLM, enedynes, and ionizing radiation. These lesioned DNAs are being used as substrates to understand the mechanism of DNA repair. The genes for the biosynthetic pathways for these natural products have been identified and the glycosylation mechanism are being examined.

Enzymes Involved in Purine Biosynthesis:
Understanding the regulation of the purine biosynthetic pathway and the importance of transient protein-protein interactions in the channeling of chemically reactive intermediates produced in this pathway. Our investigations have led to isolation and structural determination of all the enzymes in the pathway giving new insight into the evolution of a biosynthetic pathway. These studies have uncovered a new substrate and two new enzymes in this pathway!

Polyhydroxybutyrate Polymerase:
The potential for using biological systems as a source of biodegradable thermoplastics is becoming increasingly attractive given the problems associated with oil based polymers. In collaboration with the Sinskey lab in Biology, studies on the polyhydroxybutyrate polymerases, depolymerases, transcription factors and phasin proteins that govern PHB homeostasis are in progress. The mechanism of homopolymerization reactions is being used to make novel block co-polymers.

"Radical Initiation in the Class I Ribonucleotide Reductases: Long Range Electron Coupled Proton Transfer?" (2003) J. Stubbe. D. Nocera, C. Yee and M. Chang Chem Reviews in press.

"Why multiple small subunits (Y2 and Y4) for yeast ribonucleotide reductase? Toward understanding the role of Y4". Ge J, Perlstein DL, Nguyen HH, Bar G, Griffin RG, Stubbe J. Proc Natl Acad Sci U S A. 2001, 98(18):10067-72.

"Synthesis, Characterization and Solution Structure of Tethered Oligonucleotides Containing an Internal 3’-Phosphoglycolate, 5’-Phosphate Gapped Lesion" H-D. Junker, S. T. Hoehn, R. C. Bunt, C. J. Turner, J. Stubbe (2002) Nucleic Acids Research 30, 5497-508 (2002)

"The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production." York GM, Stubbe J, Sinskey AJ. J Bacteriol. 2002, 184(1):59-66.

"Mechanistic Studies on Class I Polyhydroxybutyrate (PHB) Synthase"

Ralstonia eutropha: Class I and Class III Synthases Share a Similar Catalytic Mechanism”, Y. Jia, W. Yuan, J. Wodzinska, C. Park, A. J. Sinskey, and J. Stubbe, Biochemistry, 40, 1011-1019 (2001).

"Modular Evolution of the Purine Biosynthetic Pathway", T. J. Kappock, S. E. Ealick, and J. Stubbe, Curr. Op. Chem. Biol., 4, 567-572 (2000).