Molecular Cloning of CoA Synthase

Abstract

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 ϳ60 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.

Identification, Purification, and Characterization of an Eukaryotic-like Phosphopantetheine Adenylyltransferase (Coenzyme A Biosynthetic Pathway) in the Hyperthermophilic Archaeon Pyrococcus abyssi

“…The CoA synthetic pathway has been thoroughly described in Bacteria, and more recently in Eukarya (7)(8)(9). In Archaea, of the five enzymatic steps necessary in this lower pathway starting from pantothenate, only two may be deduced with a good level of confidence from comparative genomics (Fig.…”

“…Form-Recently, the human PPAT protein domain was identified from its association with a DPCK domain and on biochemical evidence (7)(8)(9). This PPAT domain is universally present in Eukarya and it is homologous to putative archaeal nucleotidyltransferases (PACE 11), whereas it harbors no homologues in Bacteria.…”

“…The five enzymes are sequentially pantothenate kinase (EC 2.7.1.33), phosphopantothenoylcysteine synthetase (PPCS, EC 6.3.2.5), phosphopantothenoylcysteine decarboxylase (PPCDC, EC 4.1.1.36), phosphopantetheine adenylyltransferase (PPAT, EC 2.7.7.3), and dephospho-CoA kinase (DPCK, EC 2.7.1.24). The genes encoding these five activities have been identified in some bacteria such as Escherichia coli (1)(2)(3)(4)(5)(6) and in some Eukarya, such as humans (7)(8)(9). These identifications allowed a deeper biochemical characterization of the gene products (10,11).…”

“…Whereas it is common that archaeal proteins involved in cell division, metabolic or transport pathways (the so-called operational proteins) resemble more their bacterial counterparts, the reverse situation (an eukaryotic-like operational protein) is quite unusual. Putative archaeal PPAT are homologues to the recently characterized PPAT domains of the bifunctional eukaryotic PPAT/DPCK, involved in the last two steps of the coenzyme A biosynthetic pathway (7)(8)(9). We had previously characterized this archaeal protein as an interesting target for structural genomic projects (18), and listed it as a PACE (Protein of Archaea Conserved in Eukarya) protein (PACE11), with a general nucleotidyltransferase activity in our PACE data base.…”

Subcellular Localization and Regulation of Coenzyme A Synthase

“…The 4ЈPP adenylyltransferase and dephospho-CoA kinase activities of CoA synthase have been reported in the cytosol (3), the mitochondrial outer membrane (25) and in the mitochondrial matrix (26). Molecular cloning of CoA synthase cDNA has facilitated the task of analyzing subcellular localization of the enzyme and its regulation in response to various extracellular stimuli and metabolic changes (27)(28)(29).…”