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

Abstract

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.Sparsomycin (Fig. 1A) is an unusual monooxo-dithioacetalcontaining antibiotic that is produced by Streptomyces sparsogenes var. sparsogenes (3) and Streptomyces cuspidosporus (12). Sparsomycin displays bacteriocidal activity against a wide variety of gram-negative and gram-positive bacteria (27) as well as members of the Archaebacteria (17), and it exhibits potent anticancer activity against a variety of tumors in vivo and against KB human epidermoid carcinoma cells in culture (27). Sparsomycin has also been shown to potentiate the activity of other anticancer agents, such as cisplatin (25). The structure of sparsomycin was elucidated in 1970 by Wiley and MacKellar (33), while the absolute configuration was deduced by Ottenheijm and coworkers in 1981 (26). Several total syntheses of sparsomycin have been reported previously (14,20,25,26). The biological activity of sparsomycin is the result of its ability to inhibit the peptide bond-forming step of protein biosynthesis by interacting with the large ribosomal subunit in a poorly understood, complex manner (16)(17)(18)30). Although the clinical use of sparsomycin is precluded due to drug-related retinopathy, a number of more potent analogs have been prepared which may prove useful in cancer chemotherapy (16). One of the novel components of the sparsomycin molecule is (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid (UCA). Biosynthetic experiments have demonstrated (28) that this compound is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid (PCA) (Fig. 1A) and that both PCA and UCA are incorporated into sparsomycin. This article describes the first purification and characterization of an enzyme associated with the sparsomycin biosynthetic pathway. This enzyme, UCA synthase, catalyzes the NAD ϩ ...

Sparsomycin – a Review and Re-assessment

“…83 It was recognized that timing of the loss of the side chain of 75 could possibly occur earlier in the biogenetic scheme. In this regard, [6-13 C]-6-hydroxyindole (85) was not incorporated into 1, and neither were [2-13 C]-7-hydroxytryptophan (86) or [2-13 C]-7-hydroxyindole (87). The last steps in the formation of 13 were investigated with the pyrimidine derivative 88 (hypothetically derived through amination and cyclization of 84) which was labeled at the C-2 position and was well incorporated into 1.…”

“…83 Subsequently, an NAD +dependent enzyme for the conversion of (E)-3-(6-methyl-4-oxo-5-pyrimidinyl)acrylic acid (88) to 13 was purified 740-fold from S. sparsogenes. 84,85 In summary, significant progress regarding the precursor units had been made, and aspects of the overall pathways to 68 and 13 developed. However, substantial ambiguity regarding the individual pathway steps and the involvement of specific intermediates remained, although several possible intermediates had been specifically eliminated.…”

Sparsomycin Biosynthesis Highlights Unusual Module Architecture and Processing Mechanism in Non-ribosomal Peptide Synthetase

“…It was further deduced that reduction of the carboxyl group of MTM-Cys to form MTM-cysteinol precedes the attachment of UAA. Later, the same group isolated the enzyme that catalyzes the conversion of 6-methyl-pyrimidine acrylic acid to UAA, although no sequencing information was reported; interestingly, this enzyme also displays weak xanthine dehydrogenase (XDH) activity in converting hypoxanthine to xanthine. , Despite decades of biosynthetic investigations, the genetic basis for sparso­mycin biosynthesis remains unknown, which prevents the study of enzymatic reactions in transforming precursors such as l -Cys and l -Trp into the interesting compound sparso­mycin. Toward this end, here we have undertaken the identification of the sparso­mycin biosynthetic gene cluster from the producer organism S. sparsogenes through genome mining, targeted gene disruption, and heterologous expression.…”