Oxidative decarboxylation of peptides catalyzed by flavoprotein EpiD. Determination of substrate specificity using peptide libraries and neutral loss mass spectrometry.
The flavoprotein EpiD catalyzes the COOH-terminal oxidative decarboxylation of the lantibiotic precursor peptide EpiA. Variations of the COOH-terminal heptapeptide S1FNSYCC7 of EpiA were used for determining the substrate specificity of EpiD. When Cys7 was replaced by serine, cysteine-amide, homocysteine, or a thioether amino acid residue, no reaction with EpiD was observed. Heptapeptide libraries with one variable amino acid residue at positions 1-7 of the peptide substrate S1FNSYCC7 were incubated with EpiD, and the reaction products were identified by neutral loss mass spectrometry. When the penultimate cysteine residue Cys6 of the substrate peptide was replaced with Ser, Thr, Ala, or Val, the reaction still occurred. Tyr5 could be replaced with other hydrophobic amino acid residues. Mass spectrometry was used to compare the kinetics of the reaction of EpiD with various peptides. Peptide sequencing of the reaction products was performed by tandem mass spectrometry, confirming that the last cysteine residue was modified. The removal of the acid COOH-terminal carboxyl group was confirmed by determination of the isoelectric points of the reaction products. To study the interaction between EpiA and EpiD, EpiA was coupled to N-hydroxysuccinimide-activated Sepharose HiTrap material; EpiD was only retarded under reducing conditions.
Kupke
T
T
Universität Tübingen, Federal Republic of Germany.
Kempter
C
C
Jung
G
G
Götz
F
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002262:Carboxy-Lyases; D017931:DNA Primers; D005420:Flavoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010088:Oxidoreductases; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013212:Staphylococcus epidermidis / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D005420:Flavoproteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D010088:Oxidoreductases; C075971:epidermin decarboxylase; D002262:Carboxy-Lyases