PT  - JOURNAL ARTICLE
AU  - Wen-Ni Chang
AU  - Jen-Ning Tsai
AU  - Bing-Hung Chen
AU  - Huei-Sheng Huang
AU  - Tzu-Fun Fu
TI  - Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases
AID  - 10.1124/dmd.107.016840
DP  - 2007 Nov 01
TA  - Drug Metabolism and Disposition
PG  - 2127--2137
VI  - 35
IP  - 11
4099  - http://dmd.aspetjournals.org/content/35/11/2127.short
4100  - http://dmd.aspetjournals.org/content/35/11/2127.full
SO  - Drug Metab Dispos2007 Nov 01; 35
AB  - 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 high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding of the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in Escherichia 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 inhibits to a lesser extent the reaction catalyzed by the mitochondrial isozyme. This is, to our knowledge, 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. The American Society for Pharmacology and Experimental Therapeutics