Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases.
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.
Chang
Wen-Ni
WN
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan 701, Taiwan.
Tsai
Jen-Ning
JN
Chen
Bing-Hung
BH
Huang
Huei-Sheng
HS
Fu
Tzu-Fun
TF
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020869:Gene Expression Profiling; D012696:Glycine Hydroxymethyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002955:Leucovorin / Q000494:pharmacology*; D008727:Methotrexate / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; D012696:Glycine Hydroxymethyltransferase; D002955:Leucovorin; D008727:Methotrexate