Received for publication May 29, 2007.
Revised July 23, 2007.
Accepted for publication July 25, 2007.

Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin-Characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases

Abstract

Serine hydroxymethyltransferase (SHMT) provides activated one-carbon units required for the biosynthesis of nucleotides, protein and methyl group by converting serine and tetrahydrofolate to glycine and N⁵,N¹⁰-methylenetetrahydrofolate. It is postulated that SHMT activity is associated with the development of methotrexate-resistance and the in vivo activity of SHMT is regulated by the binding of N⁵-CHO-THF, the rescue agent in a high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding in the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in E.coli and purified with a three-step purification protocol. Similarities in structural, physical and kinetic properties were revealed between the recombinant zebrafish mitochondrial SHMT and its mammalian orthologs. Surprisingly, leucovorin significantly inhibits the aldol cleavage of serine catalyzed by zebrafish cytosolic SHMT but to a less extent the reaction catalyzed by the mitochondrial isozyme. This is the first report on zebrafish mitochondrial folate enzyme as well as the differential inhibition of leucovorin on these two SHMT isoforms. Western blot analysis revealed tissue-specific distribution with the highest enrichment present in liver for both cytosolic and mitochondrial SHMTs. Intracellular localization was confirmed by confocal microscopy for both mitochondrial and cytosolic SHMTs. Unexpectedly, the cytosolic isoform was observed in both nucleus and cytosol. Together with the previous report on zebrafish cytosolic SHMT, we suggest that zSHMTs can be used in in vitro assays for folate-related investigation and antifolate drug discovery.

Key words: anticancer agents, drug toxicity, enzyme inhibitors, in vitro toxicity assays, inhibition, recombinant proteins

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