| Abstract
| - Cloning and sequence analysis of the bleomycin (BLM) biosynthetic gene cluster predictedthat the two nonribosomal peptide synthetases (NRPSs), BlmIV and BlmIII, are responsible for thebiosynthesis of the BLM bithiazole moiety. BlmIV is a seven domain (C2-A2-PCP2-Cy1-A1-PCP1-Cy0)NRPS, and BlmIII is a three domain (A0-PCP0-Ox) NRPS. The three domains of Cy1-A1-PCP1 residingon the BlmIV subunit, the four domains of Cy0 residing on the BlmIV subunit, and A0-PCP0-Ox residingon the BlmIII subunit constitute the two thiazole-forming NRPS-1 and NRPS-0 modules, respectively.BlmIII-A0 was predicted to be nonfunctional, raising the question of how the NRPS-0 module activatesand loads the Cys substrate to its cognate BlmIII-PCP0. The NRPS-0 module consists of domains residingon two different subunits, requiring precise protein−protein interaction. Here, we report the productionof the BlmIV and BlmIII NRPSs as an excised domain(s), module, or intact subunit form and biochemicalcharacterizations of the resultant enzymes in vitro for their roles in BLM bithiazole biosynthesis. Ourresults (a) confirm that BlmIII-A0 is a naturally occurring nonfunctional mutant, (b) demonstrate thatBlmIV-A1 activates Cys and catalyzes both in cis aminoacylation of BlmIV-PCP1 (for NRPS-1) and intrans aminoacylation of BlmIII-PCP0 (for NRPS-0), and (c) reveal that the C-terminus of the BlmIVsubunit, characterized by the unprecedented AGHDDD(G) and PGHDDG repeats, is absolutely requiredfor in trans aminoacylation of BlmIII-PCP0. These findings underscore the flexibility and versatility ofNRPSs in both structure and mechanism for natural product biosynthesis and provide an outstandingopportunity to study the molecular recognition and protein−protein interaction mechanism in NRPSassembly line enzymology.
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