Characterization of tyrocidine synthetase 1 (TY1): requirement of posttranslational modification for peptide biosynthesis.
Tyrocidine synthetase 1 (TY1), produced by Bacillus brevis ATCC 8185, consists of a single multifunctional polypeptide chain catalyzing the activation, thioesterification, and epimerization of phenylalanine. Because we were concerned about possible posttranslational issues, a comparative study between the wild-type isolate and the in Escherichia coli overexpressed protein was performed. Analysis by matrix assisted laser desorption mass spectrometry (MALDI) provided a molecular mass of 122,516 +/- 120 Da for the recombinant protein, which is in agreement with the value of 122,590 Da calculated from the gene sequence. MALDI analysis of the tryptic fragments revealed that in the recombinant TY1 the putative 4'-phosphopantetheine binding site (562Ser) is not modified by the cofactor. The substrate specificity profiles of the amino acid dependent ATP[32P]PPi exchange reactions were identical, including activation of L-phenylserine, L-tyrosine, and L-methionine. However, the rates of the reverse adenylation reaction for the recombinant protein were only 22% relative to those of the wild-type enzyme. The aminoacylation levels of about 60% for TY1 from Bacillus brevis reduced to 1.4% in the overexpressed protein. A similar distribution of the D- and the L-isomer was detected at the thioester attachment site. The pI values of the wild-type and expressed TY1 are 4.9 and 5.0, respectively. In conclusion, it could be established that apo- and holo-TY1 differ in their amino acid activating properties. Posttranslational modification by 4'-phosphopantetheine is an essential requirement for aminoacylation, epimerization, and thus the functioning of the multienzyme in peptide synthesis.
Pfeifer
E
E
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Germany.
Pavela-Vrancic
M
M
von Döhren
H
H
Kleinkauf
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli; D005798:Genes, Bacterial; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010453:Peptide Synthases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D013379:Substrate Specificity
D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D012694:Serine; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C035180:tyrocidine synthetase