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. Be cause we were concerned about possible posttranslational issues, a com parative study between the wild-type isolate and the in Escherichia co li overexpressed protein was performed. Analysis by matrix assisted la ser 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 ((562)Ser) is not mod ified by the cofactor. The substrate specificity profiles of the amino acid dependent ATP[P-32]PPi exchange reactions were identical, includ ing activation of L-phenylserine, L-tyrosine, and L-methionine. Howeve r, the rates of the reverse adenylation reaction for the recombinant p rotein were only 22% relative to those of the wild-type enzyme. The am inoacylation 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 p i values of the wild-type and expressed TY1 are 4.9 and 5.0, respectiv ely. In conclusion, it could be established that apo- and holo-TY1 dif fer in their amino acid activating properties. Posttranslational m;odi fication by 4'-phosphopantetheine is an essential requirement for amin oacylation, epimerization, and thus the functioning of the multienzyme in peptide synthesis.