Phosphopantothenoylcysteine Synthetase from Escherichia coli

Abstract

Phosphopantothenoylcysteine synthase catalyzes the formation of (R)-4-phospho-N-pantothenoylcysteine from 4-phosphopantothenate and L-cysteine: this enzyme, involved in the biosynthesis of coenzyme A (CoA), has not previously been identified. Recently it was shown that the NH 2 -terminal domain of the Dfp protein from bacteria catalyzes the next step in CoA biosynthesis, the decarboxylation of (R)-4-phospho-N-pantothenoylcysteine to form 4-phosphopantetheine (Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838 -31846). We have partially purified phosphopantothenoylcysteine decarboxylase from Escherichia coli and demonstrated that the protein encoded by the dfp gene, here renamed coaBC, also has phosphopantothenoylcysteine synthetase activity, using CTP rather than ATP as the activating nucleoside 5-triphosphate. This discovery completes the identification of all the enzymes involved in the biosynthesis of coenzyme A in bacteria.Coenzyme A (CoA) 1 fulfils a vital role in the normal metabolism of living organisms: it has been reported that ϳ4% of all enzymes utilize either CoA, its thioesters or 4Ј-phosphopantetheine (1). As an essential cofactor of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthases, 4Ј-phosphopantetheine functions as an acyl group carrier, activating carboxylic acids for biological Claisen reactions and the formation of peptides and esters. Acetyl-CoA and succinyl-CoA are key intermediates in energy metabolism, taking part in the tricarboxylic acid cycle. It is therefore surprising that three of the CoA biosynthetic enzymes have only recently been identified in E. coli (2,3).Phosphopantothenoylcysteine decarboxylase (PPC-DC) catalyzes the decarboxylation of 4Ј-phosphopantothenoylcysteine (PPanCys) to form 4Ј-phosphopantetheine (Fig. 1). From a mechanistic perspective this reaction proves to be particularly interesting as it is unclear how the stabilization of the carbanion intermediate is achieved. To study this enzyme we have repeated, with major modifications, the published purification of PPC-DC from wild-type E. coli (4). Amino-terminal sequencing identified the dfp gene as coding for the protein having this activity. The flavin-containing Dfp protein was first identified from a temperature-sensitive mutant (dfp-707), believed to be involved in DNA and pantothenate metabolism (5, 6). However, the conditional lethality of the mutant was reported not to be a direct result of pantothenate auxotrophy, and its complete characterization was not achieved. While our work was in progress, Kupke et al. (7) also identified Dfp as having PPC-DC activity by sequence comparison with EpiD, a lantibiotic-synthesizing protein catalyzing the decarboxylation of peptidylcysteine intermediates.We now report that the protein encoded by dfp also catalyzes the formation of PPanCys from 4Ј-phosphopantothenate and L-cysteine using cytidine 5Ј-triphosphate (CTP) as the activating nucleoside 5Ј-triphosphate. The protein releases CM...

The Human Malaria Parasite Plasmodium falciparum Is Not Dependent on Host Coenzyme A Biosynthesis

“…4Ј-Phosphopantothenate was synthesized using purified recombinant Escherichia coli pantothenate kinase (PanK). 4Ј-Phosphopantothenoylcysteine and 4Ј-phosphopantetheine were a gift from Erick Strauss (Stellenbosch University, South Africa), and were synthesized as described previously (21,22). Uninfected erythrocyte extracts were also collected in 1-ml fractions for more sensitive counting in a Beckman LS 6500 Multipurpose Scintillation Counter.…”

Acyl Carrier Protein Is a Cellular Target for the Antibacterial Action of the Pantothenamide Class of Pantothenate Antimetabolites

“…Cysteine is next added to the phosphopantothenate by 4Ј-phosphopantothenoylcysteine synthase and rapidly decarboxylated to phosphopantetheine. These two steps are carried out by a bifunctional polypeptide, phosphopantothenoylcysteine synthetase decarboxylase (denoted CoaBC, formally Dfp) (2). The last two steps are carried out by two separate enzymes, namely phosphopantetheine adenylyltransferase (CoaD) (3,4) followed by the addition of the 3Ј-ribose phosphate by dephospho-CoA kinase (CoaE) (5).…”

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

“…The different 3 www.embl-heidelberg.de/ϳandrade/k2d. 4 components from the enzymatic reaction were resolved on a Supelcosil LC-18 column (50 ϫ 4.6 mm, 5 m) from Supelco protected by a pre-column filter and a Supelguard LC-18 guard column (20 ϫ 4.6 mm, 5 m).…”

“…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).…”