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Biochemistry. 2000 Aug 29;39(34):10439-47.

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

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Philipps-Universit├Ąt Marburg, Fachbereich Chemie/Biochemie, Hans-Meerwein-Strasse, 35032 Marburg, Germany.


Product assembly by nonribosomal peptide synthetases (NRPS) is initiated by starter modules that comprise an adenylation (A) and a peptidyl carrier protein (PCP) domain. Elongation modules of NRPS have in addition a condensation (C) domain that is located upstream of the A domain. They cannot initiate peptide bond formation. To understand the role of domain arrangements and the influence of the domains present upstream of the A domains of the elongation modules of TycB on the initiation and epimerization activities, we constructed a set of proteins derived from the tyrocidine synthetases of Bacillus brevis, which represent several N-terminal truncations of TycB and the 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.CATE and TycB(3)-ATE, lacking an N-terminal C domain, were capable of initiating peptide synthesis and epimerizing. In contrast, the corresponding constructs 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 provided that this reduction is due to substrate binding in an enantioselective binding pocket at the acceptor position of the C domains. By using TycB(2)(-)(3)-AT.CATE and TycB(3)-ATE, we were able to turn an elongation module into an 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 both aminoacyl-S-Ppant (TycB(3)-ATE) and peptidyl-S-Ppant (TycB(2)(-)(3)-AT.CATE) substrates, although the efficiency for epimerizing the noncognate aminoacyl-S-Ppant substrates appears to be lowered.

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