4′-Phosphopantetheine Biosynthesis in Archaea

Abstract

Coenzyme A (CoA) and 2 are essential cofactors for many enzymatic reactions, and acyl-CoA derivatives are key intermediates in energy metabolism. 4Ј-Phosphopantetheine is a cofactor of enzymes that play a role in the biosynthesis of fatty acids, polypeptide antibiotics, and polyketides. The thiol group of the cysteamine moiety of coenzyme A is the functional group because it activates substrates as thioesters.Coenzyme A is synthesized from pantothenate in five steps, and all of the genes involved in the biosynthesis of the cofactor in eubacteria, plants, and human have recently been cloned and characterized. In the first step, pantothenate is phosphorylated to 4Ј-phosphopantothenate by pantothenate kinase, which is encoded by the coaA gene. Then, (R)-4Ј-phospho-N-pantothenoylcysteine (PPC) is synthesized by the addition of cysteine to 4Ј-phosphopantothenate (CoaB activity), and in the next step, PPC is decarboxylated to 4Ј-phosphopantetheine (CoaC activity). 4Ј-Phosphopantetheine is converted to coenzyme A by the enzymes 4Ј-phosphopantetheine adenylyltransferase (CoaD) and dephospho-CoA kinase (CoaE; reviewed in Ref. 1).The key reaction in coenzyme A biosynthesis, the synthesis of the phosphopeptide-like cofactor 4Ј-phosphopantetheine from 4Ј-phosphopantothenate and cysteine, is catalyzed in Escherichia coli and in most eubacteria by the bifunctional Dfp (CoaBC) flavoprotein in a multistep process (Fig. 1) (2). 4Ј-Phosphopantothenate is activated by reaction with CTP; the 4Ј-phosphopantothenoyl-cytidylate formed is attacked by cysteine, and PPC is synthesized (2-4). Crystal structure analysis of E. coli CoaB (5) revealed the 4Ј-phosphopantothenate and CTP binding motifs of CoaB. The nucleobase binding motif II of eubacterial CTP-binding CoaBs deviates from that of eukaryotic ATP-binding PPC synthetases. In E. coli Dfp the sequence reads 307 NPDIV, whereas the sequence is 210 VPKLL in the human enzyme (residues shown in boldface are those conserved in eubacterial/eukaryotic enzymes). In the next step, PPC is oxidatively decarboxylated to 4Ј-phosphopantothenoylaminoenethiol by the NH 2 -terminal FMNbinding CoaC domain of Dfp (6 -9). Subsequent reduction of 4Ј-phosphopantothenoylaminoenethiol to 4Ј-phosphopantetheine by the reduced cofactor FMNH 2 depends on the conserved cysteine residue of the 16-amino acid 4Ј-phosphopantothenoylcysteine binding clamp, 151 PDSGSQACGDIGPGRM (6,8,9). Binding of 4Ј-phosphopantothenoylcysteine also involves the Asn residue of the PXMNXXMW motif, which contacts the carboxyl group (8, 10). The presence of conserved CoA biosynthetic genes indicates that all Archaea convert 4Ј-phosphopantothenate into coenzyme A by using CoaB, CoaC, CoaD, and CoaE activities, although cloning and functional characterization of archaebacterial coenzyme A biosynthetic genes has not been published until now (11). In this paper, we have characterized the bifunctional Dfp protein of Methanocaldococcus jannaschii. The 5Ј-coaC part and the 3Ј-coaB part of the archaebacterial dfp gene were expressed in E. coli,...

Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea

“…A number of genes that display similarity to bacterial/eukaryotic genes are present on the archaeal genomes, including those encoding PPCS, PPCDC, and PPAT. Among these, the PPCS and PPCDC from Methanocaldococcus jannaschii (13) and the PPAT from Pyrococcus abyssi (1,17) have been biochemically characterized. A striking observation was that almost all of the archaeal genomes did not harbor genes corresponding to PS and PanK.…”

A novel isoform of pantothenate synthetase in the Archaea

“…In order to detect possible adenosine nucleotide products of MM2281, standard assays containing MM2281 alone or together with E. coli PANK were carried out as described above, except that [3][4][5][6][7][8][9][10][11][12][13][14] C]b-alanine was omitted. The reaction was quenched after 180 min, and the products were cochromatographed with authentic ATP, ADP and AMP standards (Sigma).…”

“…We previously used comparative genomics to reconstruct the universal CoA biosynthetic pathway in the Bacteria, Eukaryota, and Archaea [10]. Archaeal genes for the ultimate four steps can be identified by homology in all archaeal genomes, and experimental confirmation of this assignment is available for three of these steps [11,12]. In addition, bacterial-type genes for the first two steps are obvious in a number of the nonmethanogenic Archaea.…”

Pantoate Kinase and Phosphopantothenate Synthetase, Two Novel Enzymes Necessary for CoA Biosynthesis in the Archaea

“…Sequence data indicate that the bacterial PPCS and PPCDC homologs and eukaryotic PPAT homologs are found on almost all of the archaeal genomes. The archaeal PPCS and PPCDC genes are fused in many cases, and the bifunctional protein from Methanocaldococcus jannaschii has been shown to exhibit both activities (5). The PPAT homolog from Pyrococcus abyssi has also been studied and confirmed to exhibit the expected PPAT activity (6).…”