Control of directionality in nonribosomal peptide synthesis: Role of the condensation domain in preventing misinitiation and timing of epimerization

Product assembly by nonribosomal peptide synthetases (NRPS) is initiated by starter modules that comprise an adenylation (A) and a peptidyl carrier pr otein (PCP) domain. Elongation modules of NRPS have in addition a condensat ion (C) domain that is located upstream of the A domain. They cannot initia te peptide bond formation. To understand the role of domain arrangements an d the influence of the domains present upstream of the A domains of the elo ngation modules of TycB on the initiation and epimerization activities, we constructed a set of proteins derived from the tyrocidine synthetases of Ba cillus brevis, which represent several N-terminal truncations of TycB and t he first module of TycC. The latter was fused with the thioesterase domain (Te) to give TycC(1)-CAT-Te and to ensure product turnover. TycB(2-3)-AT.CA TE and TycB(3)-ATE, lacking an N-terminal C domain, were capable of initiat ing peptide synthesis and epimerizing. In contrast, the corresponding const ructs with a cognate N-terminal C domain, TycB(2-3)-T.CATE and TycB(3)-CATE , were strongly reduced in initiation and epimerization. Evidence is also p rovided that this reduction is due to substrate binding in an enantioselect ive binding pocket at the acceptor position of the C domains. By using TycB 2-3-AT.CATE and TycB3-ATE, we were able to turn an elongation module into a n initiation module, and to establish an in-trans system for the formation of new di- and tripeptides with recombinant NRPS modules. We also show that epimerization domains of elongation modules can in principle epimerize bot h aminoacyl-S-Ppant (TycB3-ATE) and peptidyl-S-Ppant (TycB2-3-AT.CATE) subs trates, although the efficiency for epimerizing the noncognate aminoacyl-S- Ppant substrates appears to be lowered.