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 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.
Footnotes
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This work was supported by Grant NSC 95-2320-B-006-022 from the National Science Council, Taiwan.
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W.N.C. and T.F.F. contributed equally to this work.
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doi:10.1124/dmd.107.016840.
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ABBREVIATIONS: MTX, methotrexate; SHMT, serine hydroxymethyltransferase; zmSHMT and zcSHMT, zebrafish mitochondrial and zebrafish cytosolic SHMT; hcSHMT and rcSHMT, human cytosolic and rabbit cytosolic enzymes; THF or H4PteGlu, tetrahydropteroylglutamate; N5-CHO-H4PteGlu, N5-formyl-tetrahydropteroylglutamate; N5,N10-CH2-, N5,N10-methylene; N5-CH3-, N5-methyl; PLP, pyridoxal-5′-phosphate; DHFR, dihydrofolate reductase; RT-PCR, reverse transcription-polymerase chain reaction; EGFP, enhanced green fluorescent protein; IPTG, isopropyl-β-d-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; ZLE, zebrafish liver epithelial; RACE, rapid amplification of cDNA ends; UPM, Universal Primer Mix; NUP, Nested Universal Primer; bp, base pair(s); kb, kilobase(s); (F), forward; (R), reverse; GFP, green fluorescent protein; SUMO, small ubiquitin-like modifier.
- Received May 24, 2007.
- Accepted July 25, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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