Cloning, purification, and properties of Candida albicans thymidylate synthase

Abstract

The thymidylate synthase (TS) gene was isolated from a genomic Candida albicans library by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was localized on a 4-kilobase HindIll DNA fragment and was shown to be expressed in a Thy-strain of Escherichia coi. The Thymidylate synthase (TS) has been characterized from a wide variety of organisms and is a proven target in cancer chemotherapy (25). TS should be a good chemotherapeutic target in Candida albicans, a common fungal pathogen, since the product of the enzyme, dTMP, can only be synthesized de novo in yeasts; they lack thymidine kinase and are impermeable to thymine, thymidine, and dTMP (6). Effective inhibition of TS in yeasts would result in death, since these organisms are unable to produce their own dTMP or obtain it from the environment. 5-Fluorocytosine inhibits C. albicans and several other fungi both in vitro and in vivo (3). Furthermore, treatment of C. albicans with 5-fluorocytosine (9) results in 5-fluoro-dUMP accumulation and inhibition of TS, thus implicating this enzyme as a chemotherapeutic target in fungi.Information obtained from the characterization of target enzymes in vitro has contributed to the design of new potential chemotherapeutic agents. Such studies are facilitated by the availability of large quantities of pure enzyme. Since low levels of TS are present in cultures of C. albicans, the cloning and overexpression of C. albicans TS in Escherichia coli was undertaken. We report the isolation of the C. albicans TS gene by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was sequenced with about 400 base pairs (bp) of DNA 5' to the gene and a short 3'-flanking region and expressed in E. coli by using a T7 expression vector. The purified enzyme from both C. albicans and E. coli was prepared, and its properties were examined to ensure that the cloned TS enzyme as expressed in E. coli was identical to the native TS enzyme of C. albicans. MATERIALS AND METHODSStrains, plasmids, and media. isolated as a pool of 8-to 10-kilobase-pair (kb) fragments, into the BamHI site of the low-copy-number S. cerevisiae-E. coli shuttle vector pYSK35 (Fig. 1). The S. cerevisiae-E. coli shuttle vector YEp13 (8)

Isolation and expression of the Pneumocystis carinii thymidylate synthase gene.

“…The amino acid sequence shows both of the invariant eukaryote-specific insertions; a 12-amino acid loop at position 90 (between a-D and E helices) and the 8-amino acid loop after residue 157 (between a-G and H helices) of the L. casei TS. The single-amino acid insertion after residue 34 has thus far been observed only in the other fungal TSs characterized (serine in P. carinii, proline in S. cerevisiae and Candida albicans) (15,34). This insertion is found in the first 3-strand of TS, which forms an important interface for subunit contact.…”

“…Translation of the open reading frame yields a polypeptide of 297 amino acids with a molecular weight of 34,269. The deduced amino acid sequence shows conservation of all the amino acid residues that are invariant in known TSs.…”

Progress in Candida albicans Molecular Biology

“…The earliest molecular biological studies in C. albicans suggested that Candida genes were likely to resemble genes in Saccharomyces cerevisiae, since most C. albicans genes were cloned via the complementation of S. cerevisiae mutations [3,14,32,33,34,62,69]. Many C. albicans genes will express directly in Escherichia coli [14,23,34] and more recently the suspected similarity between C. albicans and S. cerevisiae genes has been confirmed by DNA sequence analysis.…”

“…The C. albicans thymidylate synthase gene was cloned by its ability to complement a tmp 1 mutation in S. cerevisiae [69]. The gene encodes a protein of 315 amino acids which, when expressed in E. coli, was enzymatically indistinguishable from the enzyme purified from C. albicans.…”

“…Thymidylate synthase has been studied as a potential target for anticandidal therapeutics [69]. It was reasoned that this enzyme should be a good target because (l) the product of the enzyme, dTMP, can be synthesized only de novo in C. albicans, since yeasts lack thymidine kinase; (2) yeasts do not take up thymine, thymidine and dTMP from the environment; and (3) 5-fiuorocytosine, a well-known anticandidal agent, acts in the cell via its conversion to 5-fiuorodUMP which is an inhibitor of thymidylate synthase.…”