Bioinorganic Chemistry; Mechanisms of Metalloenzyme-mediated Reactions; Mechanisms of Biological Radical Reactions; Bioremediation

jbroderickchemistry.montana.edu

The overall objective of this project is to delineate the detailed chemical mechanism of radical generation by the Fe/S-S-adenosylmethionine (the so-called radical SAM) superfamily of enzymes. These enzymes span a remarkably diverse range of reactions and are represented across the phylogenetic kingdom, with hundreds of radical SAM enzymes identified. The widespread occurrence of these enzymes throughout biology, from bacteria to humans, is indicative of the significance of the chemistry catalyzed by these enzymes. In humans, radical SAM enzymes are involved in the biosynthesis of lipoic acid, the synthesis of heme, and the biosynthesis of the molybdopterin cofactor, among many other essential functions, some as yet unidentified. Despite the diversity of reactions catalyzed, our overriding hypothesis is that the adenosylmethionine-dependent iron-sulfur enzymes all operate by a common mechanism in which a reduced cluster interacts with S-adenosylmethionine to generate an adenosyl radical intermediate, which is directly involved in catalysis. These reactions represent novel chemistry for iron-sulfur clusters. To investigate this novel chemistry, biochemical, spectroscopic, mechanistic, and structural studies of pyruvate formate-lyase activating enzyme (PFL-AE) are being pursued.

Selected Publications

Broderick JB :
Iron-Sulfur Clusters in Enzyme Catalysis
Comprehensive Coordination Chemistry II: From Biology to Nanotechnology, Volume 8, L. Que and W. B. Tolman, Volume Eds., J. McCleverty and T. Meyer, Eds., Elsevier Science, 2003

Krebs C, Broderick WE, Henshaw TF, Broderick JB, Huynh BH :
Coordination of Adenosylmethionine to a Unique Iron Site of the [4Fe-4S] of Pyruvate Formate-Lyase Activating Enzyme: A MÃssbauer Spectroscopic Study
J. Am. Chem. Soc. 124 912-913 (2002)

Buis JM, Broderick JB :
Pyruvate Formate-Lyase Activating Enzyme: Elucidation of a Novel Mechanism for Glycyl Radical Formation
Arch. Bioch. Biophys. 433 288-296 (2005)

Walsby C, Ortillo D, Broderick WE, Broderick JB, Hoffman BM :
An Anchoring Role for FeS Clusters: Chelation of the Amino Acid Moiety of S-Adenosylmethionine to the Unique Iron Site of the [4Fe-4S] Cluster of Pyruvate Formate-Lyase Activating Enzyme,
J. Am. Chem. Soc. 124 11270-11271 (2002)

Walsby C, Ortillo D, Yang J, Nnyepi MR, Broderick WE, Hoffman BM, Broderick JB :
Spectroscopic Approaches to Elucidating Novel Iron-Sulfur Chemistry in the "Radical SAM" Protein Superfamily
Inorg. Chem. 44 727-741 (2005)

Keywords:
Bioinorganic, Biophysical, Inorganic

Repair of UV-induced DNA damage is central to the prevention of a number of adverse conditions such as melanoma, but in most cases the mechanism of such DNA repair is not well understood at a molecular level. Under normal cellular conditions, the major DNA photoproducts of UV irradiation are cyclobutane pyrimidine dimers (TT, CT, and CC dimers) and 6,4-photoproducts, while 5-thyminyl-5,6-dihydrothymine is a minor product. This typically minor UV photoproduct becomes the major UV photoproduct under certain conditions, however, including the conditions that exist in bacterial spores (thus the common name is spore photoproduct, SP). Remarkably, the formation of spore photoproduct is correlated with the unusually high resistance of bacterial spores to UV irradiation, and this resistance arises in part from the novel DNA repair enzyme that repairs SP. The overall goals of this project include investigating the mechanism by which the repair enzyme, SP lyase, recognizes and repairs SP. In addition, we are interested in probing the mechanism by which SP is formed at the expense of cyclobutane pyrimidine dimers.

Selected Publications

Cheek J, Broderick JB :
Adenosylmethionine-Dependent Iron-Sulfur Enzymes: Versatile Clusters in a Radical New Role
J. Biol. Inorg. Chem. 6 209-226 (2001)

Cheek J, Broderick JB :
Direct H atom Abstraction from Spore Photoproduct C-6 Initiates DNA Repair in the Reaction Catalyzed by Spore Photoproduct Lyase: Evidence for a Reversibly Generated Adenosyl Radical Intermediate
J. Am. Chem. Soc. 124 2860-2862 (2002)

Keywords:
Biochemistry, Bioinorganic, Biophysical, Inorganic

Desulfitobacteria are Gram-positive, spore-forming, nitrogen fixing anaerobes with the ability to reduce many electron acceptors includint Fe(III), U(VI), Cr(VI), As(V), Mn(IV), Se(VI), NO3-, CO2, sulfite, fumarate, and humates. Furthermore, Desulfitobacteria also reductively dechlorinate aromatic and aliphatic pollutants. Importantly, most of the metals and the organochlorine reductions are coupled to ATP production and support growth providing for the organism\'s natural selection at DOE contaminant sites. The main goals of this project are 1) to gain insight into the genetic and metabolic pathways involved in dissimilatory metal reduction and reductive dechlorination, 2) to discern the commonalities among these electron-accepting processes, 3) to identify multi-protein complexes catalyzing these functions, and 4) to elucidate the coordination in expression fo these pathways and processes.

Keywords:
Biochemistry, Bioinorganic, Biophysical, Inorganic

Metalloenzymes catalyze remarkably diverse and sometimes extremely difficult reactions in biological systems. The theme of our research is the use of biochemical, spectroscopic, and synthetic approaches to elucidate detailed chemical mechanisms for some of nature's metal catalysts,

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