Molecular Characterization of Lantibiotic-synthesizing Enzyme EpiD Reveals a Function for Bacterial Dfp Proteins in Coenzyme A Biosynthesis

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. 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. (R)-4′-phospho-N-pantothenoylcysteine 4′-phosphopantetheine homododecameric flavin-containing Cys decarboxylases maltose-binding protein electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry dithiothreitol reversed phase chromatography isopropyl-1-thio-β-d-galactopyranoside polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis- (hydroxymethyl)ethyl]glycine Lantibiotics such as nisin, epidermin, mersacidin, and cytolysin are a group of ribosomally synthesized and post-translationally modified antibiotic peptides (1Schnell N. Entian K.-D. Schneider U. Götz F. Zähner H. Kellner R. Jung G. Nature. 1988; 333: 276-278Crossref PubMed Scopus (380) Google Scholar) that are produced by and act on Gram-positive bacteria. Recently, it has been shown that the antimicrobial activity of nisin arises due to its binding to the membrane-anchored cell wall precursor lipid II and subsequent permeabilization of the plasma membrane (2Breukink E. Wiedemann I. van Kraaij C. Kuipers O.P. Sahl H.-G. de Kruijff B. Science. 1999; 286: 2361-2364Crossref PubMed Scopus (622) Google Scholar).The main focus of current research is the characterization of the novel posttranslational modification reactions involved in lantibiotic biosynthesis, such as dehydration of serine and threonine residues, thioether formation, and the formation of d-alanine residues from l-serine residues (reviewed in Ref. 3Sahl H.-G. Bierbaum G. Annu. Rev. Microbiol. 1998; 52: 41-79Crossref PubMed Scopus (414) Google Scholar). Currently, only the function of flavoenzyme EpiD has been described. This enzyme binds one FMN molecule and is involved in the formation of the C-terminalS-[(Z)-2-aminovinyl]-d-cysteine residue (4Allgaier H. Jung G. Werner R.G. Schneider U. Zähner H. Angew. Chem. Int. Ed. Engl. 1985; 24: 1051-1053Crossref Scopus (87) Google Scholar) of epidermin, a lantibiotic that is produced byStaphylococcus epidermidis Tü3298.EpiD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue of epidermin precursor peptide EpiA to a (Z)-enethiol structure (5Kupke T. Stevanovic S. Sahl H.-G. Götz F. J. Bacteriol. 1992; 174: 5354-5361Crossref PubMed Google Scholar, 6Kupke T. Kempter C. Gnau V. Jung G. Götz F. J. Biol. Chem. 1994; 269: 5653-5659Abstract Full Text PDF PubMed Google Scholar, 7Kupke T. Kempter C. Jung G. Götz F. J. Biol. Chem. 1995; 270: 11282-11289Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Two reducing equivalents from the C-terminal cysteine residue of EpiA are removed; a double bond is formed; the coenzyme FMN is reduced to FMNH2, and then the C-terminal carboxyl group is removed. The unusual enethiol structure has been confirmed by two-dimensional NMR spectroscopy (8Kempter C. Kupke T. Kaiser D. Metzger J.W. Jung G. Angew. Chem. Int. Ed. Engl. 1996; 35: 2104-2107Crossref Scopus (24) Google Scholar). The pK a of the enethiol group is 6.0 (9Kupke T. Götz F. J. Biol. Chem. 1997; 272: 4759-4762Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), indicating that the enethiol group is far more reactive than the thiol group of cysteine residues at physiological pH values. The formation of theS-[(Z)-2-aminovinyl]-d-cysteine structure can then be explained by the addition of the thiol group of the C-terminal enethiol to didehydroalanine at position 19 formed by the dehydration of a serine residue. EpiD has a wide substrate specificity, and most of the peptides with the sequence (V/I/L/(M)/F/Y/W)-(A/S/V/T/C/(I/L))-C at the C terminus are substrates of EpiD, as elucidated by analysis of the reaction of EpiD with single peptides and peptide libraries (7Kupke T. Kempter C. Jung G. Götz F. J. Biol. Chem. 1995; 270: 11282-11289Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar).A decarboxylation reaction of a cysteine residue also occurs in the pathway for the biosynthesis of coenzyme A. Coenzyme A is required for synthetic and degradative reactions in intermediary metabolism and is the principal acyl group carrier in all living cells (10Abiko Y. Greenburg D.M. Metabolic Pathways. Academic Press, Inc., New York1975: 1-25Google Scholar). The synthesis of coenzyme A is a multiple step process. β-Alanine is synthesized from l-aspartate by the panD gene product, an aspartate-1-decarboxylase. Then β-alanine combines with pantoate to form pantothenate. This is converted in a two-step process in bacteria to (R)-4′-phospho-N-pantothenoylcysteine (PPC)1 (11Jackowski S. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 687-694Google Scholar). The cysteine residue of PPC is then decarboxylated to the reactive cysteamine residue of 4′-phosphopantetheine (PP), a reaction that was attributed to a pyruvoyl-dependent enzyme (12Yang H. Abeles R.H. Biochemistry. 1987; 26: 4076-4081Crossref PubMed Scopus (22) Google Scholar). PP is the precursor of coenzyme A and the prosthetic group of the acyl carrier protein. PP is converted in two steps to coenzyme A by the enzymes phosphopantetheine adenylyltransferase and dephospho-CoA kinase (11Jackowski S. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 687-694Google Scholar).In this study, we characterized the molecular basis of the action of flavoenzyme EpiD in order to elucidate the function of homologous proteins, which are present in all kingdoms of life. This work first led to identification of a new flavin coenzyme binding motif and to identification of the active-site residues of EpiD. By homology we proposed that the Dfp proteins from eubacteria and archaea also catalyze a decarboxylation reaction and/or have peptidylcysteines as substrates. Based on the obtained data for EpiD, we then elucidated the previously unknown function of bacterial Dfp proteins, and we demonstrated that these flavoenzymes catalyze the decarboxylation of (R)-4′-phospho-N-pantothenoylcysteine to 4′-phosphopantetheine. We also present data that indicate that EpiD, Dfp, and all other homologous proteins are homododecamers, and therefore we introduce the name HFCD (homododecamericflavin-containing Cysdecarboxylases) proteins for them. Lantibiotics such as nisin, epidermin, mersacidin, and cytolysin are a group of ribosomally synthesized and post-translationally modified antibiotic peptides (1Schnell N. Entian K.-D. Schneider U. Götz F. Zähner H. Kellner R. Jung G. Nature. 1988; 333: 276-278Crossref PubMed Scopus (380) Google Scholar) that are produced by and act on Gram-positive bacteria. Recently, it has been shown that the antimicrobial activity of nisin arises due to its binding to the membrane-anchored cell wall precursor lipid II and subsequent permeabilization of the plasma membrane (2Breukink E. Wiedemann I. van Kraaij C. Kuipers O.P. Sahl H.-G. de Kruijff B. Science. 1999; 286: 2361-2364Crossref PubMed Scopus (622) Google Scholar). The main focus of current research is the characterization of the novel posttranslational modification reactions involved in lantibiotic biosynthesis, such as dehydration of serine and threonine residues, thioether formation, and the formation of d-alanine residues from l-serine residues (reviewed in Ref. 3Sahl H.-G. Bierbaum G. Annu. Rev. Microbiol. 1998; 52: 41-79Crossref PubMed Scopus (414) Google Scholar). Currently, only the function of flavoenzyme EpiD has been described. This enzyme binds one FMN molecule and is involved in the formation of the C-terminalS-[(Z)-2-aminovinyl]-d-cysteine residue (4Allgaier H. Jung G. Werner R.G. Schneider U. Zähner H. Angew. Chem. Int. Ed. Engl. 1985; 24: 1051-1053Crossref Scopus (87) Google Scholar) of epidermin, a lantibiotic that is produced byStaphylococcus epidermidis Tü3298. EpiD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue of epidermin precursor peptide EpiA to a (Z)-enethiol structure (5Kupke T. Stevanovic S. Sahl H.-G. Götz F. J. Bacteriol. 1992; 174: 5354-5361Crossref PubMed Google Scholar, 6Kupke T. Kempter C. Gnau V. Jung G. Götz F. J. Biol. Chem. 1994; 269: 5653-5659Abstract Full Text PDF PubMed Google Scholar, 7Kupke T. Kempter C. Jung G. Götz F. J. Biol. Chem. 1995; 270: 11282-11289Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Two reducing equivalents from the C-terminal cysteine residue of EpiA are removed; a double bond is formed; the coenzyme FMN is reduced to FMNH2, and then the C-terminal carboxyl group is removed. The unusual enethiol structure has been confirmed by two-dimensional NMR spectroscopy (8Kempter C. Kupke T. Kaiser D. Metzger J.W. Jung G. Angew. Chem. Int. Ed. Engl. 1996; 35: 2104-2107Crossref Scopus (24) Google Scholar). The pK a of the enethiol group is 6.0 (9Kupke T. Götz F. J. Biol. Chem. 1997; 272: 4759-4762Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), indicating that the enethiol group is far more reactive than the thiol group of cysteine residues at physiological pH values. The formation of theS-[(Z)-2-aminovinyl]-d-cysteine structure can then be explained by the addition of the thiol group of the C-terminal enethiol to didehydroalanine at position 19 formed by the dehydration of a serine residue. EpiD has a wide substrate specificity, and most of the peptides with the sequence (V/I/L/(M)/F/Y/W)-(A/S/V/T/C/(I/L))-C at the C terminus are substrates of EpiD, as elucidated by analysis of the reaction of EpiD with single peptides and peptide libraries (7Kupke T. Kempter C. Jung G. Götz F. J. Biol. Chem. 1995; 270: 11282-11289Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). A decarboxylation reaction of a cysteine residue also occurs in the pathway for the biosynthesis of coenzyme A. Coenzyme A is required for synthetic and degradative reactions in intermediary metabolism and is the principal acyl group carrier in all living cells (10Abiko Y. Greenburg D.M. Metabolic Pathways. Academic Press, Inc., New York1975: 1-25Google Scholar). The synthesis of coenzyme A is a multiple step process. β-Alanine is synthesized from l-aspartate by the panD gene product, an aspartate-1-decarboxylase. Then β-alanine combines with pantoate to form pantothenate. This is converted in a two-step process in bacteria to (R)-4′-phospho-N-pantothenoylcysteine (PPC)1 (11Jackowski S. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 687-694Google Scholar). The cysteine residue of PPC is then decarboxylated to the reactive cysteamine residue of 4′-phosphopantetheine (PP), a reaction that was attributed to a pyruvoyl-dependent enzyme (12Yang H. Abeles R.H. Biochemistry. 1987; 26: 4076-4081Crossref PubMed Scopus (22) Google Scholar). PP is the precursor of coenzyme A and the prosthetic group of the acyl carrier protein. PP is converted in two steps to coenzyme A by the enzymes phosphopantetheine adenylyltransferase and dephospho-CoA kinase (11Jackowski S. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 687-694Google Scholar). In this study, we characterized the molecular basis of the action of flavoenzyme EpiD in order to elucidate the function of homologous proteins, which are present in all kingdoms of life. This work first led to identification of a new flavin coenzyme binding motif and to identification of the active-site residues of EpiD. By homology we proposed that the Dfp proteins from eubacteria and archaea also catalyze a decarboxylation reaction and/or have peptidylcysteines as substrates. Based on the obtained data for EpiD, we then elucidated the previously unknown function of bacterial Dfp proteins, and we demonstrated that these flavoenzymes catalyze the decarboxylation of (R)-4′-phospho-N-pantothenoylcysteine to 4′-phosphopantetheine. We also present data that indicate that EpiD, Dfp, and all other homologous proteins are homododecamers, and therefore we introduce the name HFCD (homododecamericflavin-containing Cysdecarboxylases) proteins for them. We thank Regine Stemmler for excellent technical assistance, Stefan Stevanovic for N-terminal sequencing of Dfp, and Lloyd Ruddock for editing the manuscript. We thank Karsten Altena and Gabriele Bierbaum for providing the MrsD sequence prior its publication.