Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin.
Sparsomycin is an antitumor antibiotic produced by Streptomyces sparsogenes. Biosynthetic experiments have previously demonstrated that one component of sparsomycin is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid and (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid. An enzyme which catalyzes the conversion of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid to (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid has been purified 740-fold to homogeneity from S. sparsogenes. The molecular mass of the native and denatured enzyme was 87 kDa, indicating that the native enzyme is monomeric. The enzyme required NAD+ for activity but lacked rigid substrate specificity, since analogs of both NAD+ and 3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid could serve as substrates. The enzyme was very weakly inhibited by mycophenolic acid. Monovalent cations were required for activity, with potassium ions being the most effective. The enzyme exhibited sensitivity toward diethylpyrocarbonate and some thiol-directed reagents, and it was irreversibly inhibited by 6-chloropurine. The properties of the enzyme suggest it is mechanistically related to inosine-5'-monophosphate dehydrogenase.
Parry
R J
RJ
Department of Chemistry, Rice University, Houston, Texas 77251, USA. parry@rice.edu
Hoyt
J C
JC
D000179:Acrylates / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis*; D002412:Cations / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D007042:Hypoxanthines / Q000494:pharmacology; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009173:Mycophenolic Acid / Q000494:pharmacology; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology; D011744:Pyrimidinones / Q000378:metabolism*; D013033:Sparsomycin / Q000096:biosynthesis*; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000179:Acrylates; D000596:Amino Acids; D000903:Antibiotics, Antineoplastic; D002412:Cations; D007042:Hypoxanthines; D011687:Purines; D011744:Pyrimidinones; D013439:Sulfhydryl Reagents; D009243:NAD; C104243:2-chlorohypoxanthine; C104242:3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid; D013033:Sparsomycin; D010105:Oxygenases; C104244:3-(2,4--dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase; D009173:Mycophenolic Acid; D004047:Diethyl Pyrocarbonate; C079308:6-chloropurine