Complete Reconstitution of the Human Coenzyme A Biosynthetic Pathway via Comparative Genomics

Abstract

The biosynthesis of CoA from pantothenic acid (vitamin B 5 ) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC 6.3.2.5), phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36), phosphopantetheine adenylyltransferase (EC 2.7.7.3), and dephospho-CoA kinase (EC 2.7.1.24). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.

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).…”

“…In silico searches in completely sequenced archaeal genomes using known bacterial and eukaryal proteins involved in the CoA biosynthetic pathway allowed identification of archaeal homologues for only three of the five steps, PPCS, PPCD, and PPAT. Putative archaeal PPCS and PPCDC are homologous to their bacterial counterparts (a putative distantly related eukaryal PPCDC homologue was recently identified with a genomic approach, whereas no PPCS homologue could be highlighted (8)), whereas putative archaeal PPAT are only homologous to their eukaryal counterparts. 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.…”

“…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.…”