The ribonucleotide reductase system, consisting of ribonucleotide reductase, thioredoxin reductase and thioredoxin, catalyzes the irreversible reduction of ribonucleotides to 2' deoxyribonucleotides. This reaction provides the deoxyribo-nucleotide precursors necessary for DNA synthesis and thus controls the S phase of the cell cycle and cell proliferation. It is not known how many different types of reductases occur in the different phyla and the exact mechanism of ribonucleotide reduction has not yet been elucidated. Thus far, three different ribonucleotide reductases have been recognized: prototype. This enzyme contains a stable tyrosyl of which the E. coli enzyme is the prototype. This enzyme contains a stable tyrosyl radical which renders the enzyme sensitive to hydroxylamines. Treatment of the enzyme with hydroxyurea eliminates the radical and abolishes enzyme activity. Clinically, hydroxyurea is used in the treatment of adult leukemias. 2) Coryneform bacteria utilize a recently discovered manganese-containing reductase, which may also involve a stable organic radical. 3) Many procaryotes contain a deoxyadenosylcobalamin-dependent reductase. Because these reductases do not contain a free radical, they are not inactivated by hydroxyurea. Recent evidence has indicated that anaerobic conditions give rise to yet another type of reductase(s). Early assumptions that anaerobes use the adenosylcobalamin-dependent enzyme have been proven to be incorrect. We have provided convincing evidence that the strictly anaerobic methananogen Methanobacterium thermoautotrophicum does not utilize adenosylcobalamin as a coenzyme. Since O2 is required in the enzymatic formation of the tyrosyl radical in the non-heme iron reductases, a different radical generating system must function in anaerobes. Our proposed studies are directed towards: a) the isolation and characterization of ribonucleotide reductase from M. thermoautotrophicum; b) the cloning and characterization of (a) thioredoxin(s) from this strict anaerobe; c) the effect of O2 on the expression of thioredoxins in Corynebacterium nephridii; d) the characterization of the active site of the adenosylcobalamin-dependent ribonucleotide reductase from C. nephridii.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033776-09
Application #
3283761
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-07-01
Project End
1994-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
9
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Krone, U E; McFarlan, S C; Hogenkamp, H P (1994) Purification and partial characterization of a putative thymidylate synthase from Methanobacterium thermoautotrophicum. Eur J Biochem 220:789-94
Loida, P J; Sligar, S G (1993) Molecular recognition in cytochrome P-450: mechanism for the control of uncoupling reactions. Biochemistry 32:11530-8
Ong, S P; McFarlan, S C; Hogenkamp, H P (1993) 2'-C-methyladenosine and 2'-C-methyluridine 5'-diphosphates are mechanism-based inhibitors of ribonucleoside diphosphate reductase from Corynebacterium nephridii. Biochemistry 32:11397-404
Ong, S P; Nelson, L S; Hogenkamp, H P (1992) Synthesis of 3'-C-methyladenosine and 3'-C-methyluridine diphosphates and their interaction with the ribonucleoside diphosphate reductase from Corynebacterium nephridii. Biochemistry 31:11210-5
Schallreuter, K U; Elgren, T E; Nelson Jr, L S et al. (1992) Ribonucleotide diphosphate reductase from human metastatic melanoma. Melanoma Res 2:393-400
McFarlan, S C; Terrell, C A; Hogenkamp, H P (1992) The purification, characterization, and primary structure of a small redox protein from Methanobacterium thermoautotrophicum, an archaebacterium. J Biol Chem 267:10561-9
Sze, I S; McFarlan, S C; Spormann, A et al. (1992) A possible new class of ribonucleotide reductase from Methanobacterium thermoautotrophicum. Biochem Biophys Res Commun 184:1101-7
McFarlan, S C; Hogenkamp, H P; Eccleston, E D et al. (1989) Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii. Eur J Biochem 179:389-98
Lim, C J; Fuchs, J A; McFarlan, S C et al. (1987) Cloning, expression, and nucleotide sequence of a gene encoding a second thioredoxin from Corynebacterium nephridii. J Biol Chem 262:12114-9
Hogenkamp, H P; Bratt, G T; Kotchevar, A T (1987) Reaction of alkylcobalamins with thiols. Biochemistry 26:4723-7

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