pantothenylcysteine-4--phosphate and 4--phosphopantetheine

Phosphopantothenoylcysteine decarboxylase (PPC-DC) catalyzes the decarboxylation of the cysteine moiety of 4'-phosphopantothenoylcysteine (PPC) to form 4'-phosphopantetheine (PPantSH); this reaction forms part of the biosynthesis of coenzyme A. The enzyme is a member of the larger family of cysteine decarboxylases including the lantibiotic-biosynthesizing enzymes EpiD and MrsD, all of which use a tightly bound flavin cofactor to oxidize the thiol moiety of the substrate to a thioaldehyde. The thioaldehyde serves to delocalize the charge that develops in the subsequent decarboxylation reaction. In the case of PPC-DC enzymes the resulting enethiol is reduced to a thiol giving net decarboxylation of cysteine, while in EpiD and MrsD it is released as the final product of the reaction. In this paper, we describe the characterization of the novel cyclopropyl-substituted product analogue 4'-phospho-N-(1-mercaptomethyl-cyclopropyl)-pantothenamide (PPanDeltaSH) as a mechanism-based inhibitor of the human PPC-DC enzyme. This inhibitor alkylates the enzyme on Cys(173), resulting in the trapping of a covalently bound enethiolate intermediate. When Cys(173) is exchanged for the weaker acid serine by site-directed mutagenesis the enethiolate reaction intermediate also accumulates. This suggests that Cys(173) serves as an active site acid in the protonation of the enethiolate intermediate in PPC-DC enzymes. We propose that this protonation step is the key mechanistic difference between the oxidative decarboxylases EpiD and MrsD (which have either serine or threonine at the corresponding position in their active sites) and PPC-DC enzymes, which also reduce the intermediate in an overall simple decarboxylation reaction.

Topics: Binding Sites; Carboxy-Lyases; Catalysis; Cysteine; Decarboxylation; Enzyme Inhibitors; Enzyme Stability; Flavin Mononucleotide; Humans; Kinetics; Mutagenesis, Site-Directed; Pantetheine; Pantothenic Acid; Serine; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity; Sulfhydryl Compounds

The Arabidopsis thaliana flavoprotein AtHAL3a is related to plant growth and salt and osmotic tolerance. AtHAL3a shows sequence homology to the bacterial flavoproteins EpiD and Dfp. EpiD, Dfp, and AtHAL3a are members of the homo-oligomeric flavin-containing Cys decarboxylase (HFCD) protein family. We demonstrate that AtHAL3a catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine. This key step in coenzyme A biosynthesis is catalyzed in bacteria by the Dfp proteins. Exchange of His-90 of AtHAL3a for Asn led to complete inactivation of the enzyme. Dfp and AtHAL3a are characterized by a shortened substrate binding clamp compared with EpiD. Exchange of the cysteine residue of the conserved ACGD motif of this binding clamp resulted in loss of (R)-4'-phospho-N-pantothenoylcysteine decarboxylase activity. Based on the crystal structures of EpiD H67N with bound substrate peptide and of AtHAL3a, we present a model for the binding of (R)-4'-phospho-N-pantothenoylcysteine to AtHAL3a.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Binding Sites; Carboxy-Lyases; Catalysis; Chromatography, Gel; Coenzyme A; Crystallography, X-Ray; Cysteine; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Models, Chemical; Models, Molecular; Molecular Sequence Data; Multienzyme Complexes; Mutagenesis, Site-Directed; Mutation; Oxidoreductases; Pantetheine; Pantothenic Acid; Peptide Synthases; Plant Proteins; Polymerase Chain Reaction; Protein Binding; Protein Conformation; Salts; Sequence Homology, Amino Acid; Signal Transduction; Time Factors

Topics: Carboxy-Lyases; Catalysis; Coenzyme A; Cysteine; Cytidine Monophosphate; Cytidine Triphosphate; Enzyme Activation; Escherichia coli; Molecular Structure; Pantetheine; Pantothenic Acid; Peptide Synthases; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity

The lantibiotic-synthesizing flavoprotein EpiD catalyzes the oxidative decarboxylation of peptidylcysteines to peptidyl-aminoenethiols. The sequence motif responsible for flavin coenzyme binding and enzyme activity is conserved in different proteins from all kingdoms of life. Dfp proteins of eubacteria and archaebacteria and salt tolerance proteins of yeasts and plants belong to this new family of flavoproteins. The enzymatic function of all these proteins was not known, but our experiments suggested that they catalyze a similar reaction like EpiD and/or may have similar substrates and are homododecameric flavoproteins. We demonstrate that the N-terminal domain of the Escherichia coli Dfp protein catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine. This reaction is essential for coenzyme A biosynthesis.

Topics: Amino Acid Motifs; Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Carboxy-Lyases; Chromatography, Gel; Coenzyme A; Cysteine; Escherichia coli; Flavin Mononucleotide; Flavoproteins; Molecular Sequence Data; Mutation; Oxidoreductases; Pantetheine; Pantothenic Acid; Peptides; Protein Binding; Protein Conformation; Recombinant Fusion Proteins; Sequence Homology, Amino Acid