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 sparso genes. Biosynthetic experiments have previously demonstrated that one component of sparsomycin is derived from L-tryptophan via the intermed iacy 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 purifie d 740-fold to homogeneity from S. sparsogenes, The molecular mass of t he native and denatured enzyme was 87 kDa, indicating that the native enzyme is monomeric, The enzyme required NAD(+) for activity but lacke d rigid substrate specificity, since analogs of both NAD(+) and 3-(4-o xo-6-methyl-5-pyrimidinyl)acrylic acid could serve as substrates. The enzyme was very weakly inhibited by mycophenolic acid. Monovalent cati ons were required for activity, with potassium ions being the most eff ective, The enzyme exhibited sensitivity toward diethylpyrocarbonate a nd some thiol-directed reagents, and it was irreversibly inhibited by 6-chloropurine, The properties of the enzyme suggest it is mechanistic ally related to inosine-5'-monophosphate dehydrogenase.