BlmIII and BlmIV nonribosomal peptide synthetase-catalyzed biosynthesis of the bleomycin bithiazole moiety involving both in cis and in trans aminoacylation

Liangcheng Du, Mei Chen, Yang Zhang, Ben Shen

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

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

Original languageEnglish (US)
Pages (from-to)9731-9740
Number of pages10
JournalBiochemistry
Volume42
Issue number32
DOIs
StatePublished - Aug 19 2003

ASJC Scopus subject areas

  • Biochemistry

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