Purification and Characterization of Phosphopantetheine Adenylyltransferase from Escherichia coli

Abstract

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4-phosphopantetheine yielding 3-dephospho-CoA and pyrophosphate. Wild-type PPAT from Escherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamed coaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.Coenzyme A (CoA) 1 is an essential cofactor in numerous biosynthetic, degradative, and energy-yielding metabolic pathways and is involved in the control of several key reactions in intermediary metabolism (1). CoA also donates the 4Ј-phosphopantetheinyl cofactor to the acyl carrier protein of the fatty acid synthase complex (2).The synthesis of CoA occurs in five steps which, utilize pantothenate (vitamin B 5 ), cysteine, and ATP (for review, see Ref.3). In all systems studied, the rate of CoA biosynthesis appears to be regulated by feedback inhibition of the first enzyme of the pathway, pantothenate kinase (4 -7). In vitro studies of pantothenate kinase from Escherichia coli showed that (a) CoA and, to a lesser extent, its acyl thioesters are competitive inhibitors with respect to ATP and (b) the K i values are within the physiological range of intracellular CoA concentrations (6). Studies of the intermediates in CoA biosynthesis have shown that both pantothenate and 4Ј-phosphopantetheine can accumulate in the cell (8). Hence, in addition to control of CoA synthesis on the level of pantothenate kinase, further modulation of flux through the pathway could occur at phosphopantetheine adenylyltransferase (PPAT), which catalyzes the penultimate step in the pathway (Fig. 1), the reversible adenylation of 4Ј-phosphopantetheine to form 3Ј-dephospho-CoA (dPCoA) and pyrophosphate (PP i ). Regulation at this step may control the reutilization of 4Ј-phosphopantetheine arising either from the turnover of the 4Ј-phosphopantetheinyl cofactor of the acyl carrier protein (8) or the cleavage of CoA by a phosphodiesterase (9).Despite the above arguments for a role in the regulation of CoA biosynthesis, PPAT has not been the subject of a detailed study. Enzymes with PPAT activity have been purified from a number of different organisms. In mammals PPAT has been shown to be part of a complex that also includes dPCoA kinase, the effector of the fina...

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

“…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). E. coli is capable of de novo pantothenate biosynthesis (1) or can import pantothenate from the medium via a sodium-dependent active transport process (6)(7)(8).…”

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

“…Geerlof et al (5) have shown that homologous overexpression of an enterobacterial coaD gene in E. coli led to the production of a protein containing a CoA molecule. The UV spectrum measured for His-tagged PAB0944 is reported in the inset of Fig.…”

“…Secondary structure predictions were obtained through the PSIpred v2.4 web-interfaced facilities 4 described by McGuffin et al (20) or through the IBPC consensus program. 5 Nuclear Magnetic Resonance Spectroscopy-Unlabeled PAB0944 sample was prepared at a concentration of 0.1 mM in a 10 mM KH 2 PO 4 / Na 2 HPO 4 buffer (pH 7.2), and 10% D 2 O. The sample was degassed, argon-saturated, and sealed before analysis.…”

“…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).…”

Three Enzymatic Steps Required for the Galactosamine Incorporation into Core Lipopolysaccharide

“…The 3Ј-end of the cluster contains the waaA (CMP-Kdo:lipidA Kdo bifunctional transferase and waaE (glucosyltransferase) genes adjacent to core OS-unrelated genes coaD (phosphopantetheine adenylyltransferase) (29) and fpg (formamidopyrimidine-DNA glycosylase) (30). In the middle of the cluster, genes waaQ (ADP-L-glycero-␣-Dmanno-heptosepyranose transferase III), wamA (ADP-D-glycero-D-manno-heptopyranose transferase), wabG (UDP-GalA transferase), wabH-like (UDP-GlcNAc transferase), and wabN (LPS-GlcNAc deacetylase) were identified (Fig.…”