Enzymatic properties of ALDH1L2, a mitochondrial 10-formyltetrahydrofolate dehydrogenase.
10-Formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1), an abundant cytosolic enzyme of folate metabolism, shares significant sequence similarity with enzymes of the aldehyde dehydrogenase (ALDH) family. The enzyme converts 10-formyltetrahydrofolate (10-fTHF) to tetrahydrofolate and CO(2) in an NADP(+)-dependent manner. The mechanism of this reaction includes three consecutive steps with the final occurring in an ALDH-homologous domain. We have recently identified a mitochondrial isoform of FDH (mtFDH), which is the product of a separate gene, ALDH1L2. Its overall identity to cytosolic FDH is about 74%, and the identity between the ALDH domains rises up to 79%. In the present study, human mtFDH was expressed in Escherichia coli, purified to homogeneity, and characterized. While the recombinant enzyme was capable of catalyzing the 10-fTHF hydrolase reaction, it did not produce detectable levels of ALDH activity. Despite the lack of typical ALDH catalysis, mtFDH was able to perform the characteristic 10-fTHF dehydrogenase reaction after reactivation by recombinant 4'-phosphopantetheinyl transferase (PPT) in the presence of coenzyme A. Using site-directed mutagenesis, it was determined that PPT modifies mtFDH specifically at Ser375. The C-terminal domain of mtFDH (residues 413-923) was also expressed in E. coli and characterized. This domain was found to exist as a tetramer and to catalyze an esterase reaction that is typical of other ALDH enzymes. Taken together, our studies suggest that ALDH1L2 has enzymatic properties similar to its cytosolic counterpart, although the inability to catalyze the ALDH reaction with short-chain aldehyde substrates remains an unresolved issue at present.
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, United States.
Kyle C
KC
Strickland
Natalia I
NI
Krupenko
Marianne E
ME
Dubard
Calvin J
CJ
Hu
Yaroslav
Y
Tsybovsky
Sergey A
SA
Krupenko
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D002457:Cell Extracts; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D000099295:Formyltetrahydrofolate Dehydrogenase
D001426:Bacterial Proteins; D002457:Cell Extracts; D004950:Esterases; D006867:Hydrolases; D007527:Isoenzymes; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011994:Recombinant Proteins; D012694:Serine; D017855:Transferases (Other Substituted Phosphate Groups); D000099295:Formyltetrahydrofolate Dehydrogenase; C082662:phosphopantetheinyl transferase
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