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Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases.
Drug Metab Dispos. 2007 Nov; 35(11):2127-37.DM

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 N(5),N(10)-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 N(5)-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.

Authors+Show Affiliations

Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17664250

Citation

Chang, Wen-Ni, et al. "Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited By Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 35, no. 11, 2007, pp. 2127-37.
Chang WN, Tsai JN, Chen BH, et al. Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases. Drug Metab Dispos. 2007;35(11):2127-37.
Chang, W. N., Tsai, J. N., Chen, B. H., Huang, H. S., & Fu, T. F. (2007). Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 35(11), 2127-37.
Chang WN, et al. Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited By Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases. Drug Metab Dispos. 2007;35(11):2127-37. PubMed PMID: 17664250.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases. AU - Chang,Wen-Ni, AU - Tsai,Jen-Ning, AU - Chen,Bing-Hung, AU - Huang,Huei-Sheng, AU - Fu,Tzu-Fun, Y1 - 2007/07/30/ PY - 2007/8/1/pubmed PY - 2008/1/18/medline PY - 2007/8/1/entrez SP - 2127 EP - 37 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab. Dispos. VL - 35 IS - 11 N2 - 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 N(5),N(10)-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 N(5)-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. SN - 0090-9556 UR - https://www.unboundmedicine.com/medline/citation/17664250/Serine_hydroxymethyltransferase_isoforms_are_differentially_inhibited_by_leucovorin:_characterization_and_comparison_of_recombinant_zebrafish_serine_hydroxymethyltransferases_ L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&pmid=17664250 DB - PRIME DP - Unbound Medicine ER -