Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis.
Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.
Zhyvoloup
Alexander
A
Department of Structure and Function of Nucleic Acid, The Institute of Molecular Biology and Genetics, 150 Zabolotnogo Street, Kyiv 143, Ukraine.
Nemazanyy
Ivan
I
Babich
Aleksei
A
Panasyuk
Ganna
G
Pobigailo
Natalya
N
Vudmaska
Mariya
M
Naidenov
Valeriy
V
Kukharenko
Oleksandr
O
Palchevskii
Sergiy
S
Savinska
Liliya
L
Ovcharenko
Galina
G
Verdier
Frederique
F
Valovka
Taras
T
Fenton
Tim
T
Rebholz
Heike
H
Wang
Mong-Lien
ML
Shepherd
Peter
P
Matsuka
Genadiy
G
Filonenko
Valeriy
V
Gout
Ivan T
IT
D000106:Acetate-CoA Ligase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014018:Tissue Distribution; D014166:Transferases / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D014166:Transferases; C509687:COASY protein, human; D000106:Acetate-CoA Ligase; D003065:Coenzyme A