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Epidermin from Staphylococcus epidermidis Tu È 3298 is an antimicrobial peptide of the lantibiotic family that contains, amongst other unusual amino acids, S-[(Z)-2-aminovinyl]-D-cysteine. This residue is introduced by post-translational modi®cation of the ribosomally synthesized precursor EpiA. Modi®cation starts with the oxidative decarboxylation of its C-terminal cysteine by the¯avoprotein EpiD generating a reactive (Z)-enethiol intermediate. We have determined the crystal structures of EpiD and EpiD H67N in complex with the substrate pentapeptide DSYTC at 2.5 A Ê resolution. Rossmann-type monomers build up a dodecamer of 23 point symmetry with trimers disposed at the vertices of a tetrahedron. Oligomer formation is essential for binding of¯avin mononucleotide and substrate, which is buried by an otherwise disordered substrate recognition clamp. A pocket for the tyrosine residue of the substrate peptide is formed by an induced ®t mechanism. The substrate contacts¯avin mononucleotide only via Cys-Sg, suggesting its oxidation as the initial step. A thioaldehyde intermediate could undergo spontaneous decarboxylation. The unusual substrate recognition mode and the type of chemical reaction performed provide insight into a novel family of¯avoproteins.

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 Nterminal domain of the Escherichia coli Dfp protein catalyzes the decarboxylation of (R)-4-phospho-Npantothenoylcysteine to 4-phosphopantetheine. This reaction is essential for coenzyme A biosynthesis.Lantibiotics such as nisin, epidermin, mersacidin, and cytolysin are a group of ribosomally synthesized and post-translationally modified antibiotic peptides (1) 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 (2).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 Dalanine residues from L-serine residues (reviewed in Ref. EpiD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue of epidermin precursor peptide EpiA to a (Z)-enethiol structure (5-7). Two reducing equivalents from the C-terminal cysteine residue of EpiA are removed; a double bond is formed; the coenzyme FMN is reduced to FMNH 2 , and then the C-terminal carboxyl group is removed. The unusual enethiol structure has been confirmed by two-dimensional NMR spectroscopy (8). The pK a of the enethiol group is 6.0 (9), indicating that the enethiol group is far more reactive than the thiol group of cysteine residues at physiological pH values. The formation of the S-[(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

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