Cloning, sequencing, and biochemical characterization of the nostocyclopeptide biosynthetic gene cluster: molecular basis for imine macrocyclization.

Nostocyclopeptides A1 and A2 are novel cyclic heptapeptides produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789 that possess a unique imino linkage in the macrocyclic ring. Herein we report the cloning, sequencing, annotation, and biochemical analysis of the 33-kb nostocyclopeptide (ncp) biosynthetic gene cluster, which includes seven open reading frames predicted to be involved in the biosynthesis and transport of these natural products. The genetic architecture and domain organization of the ncpA-B nonribosomal peptide synthetase (NRPS) is co-linear in arrangement with respect to the putative order of the biosynthetic assembly of the cyclic peptide. A reductase domain identified at the C-terminal end of the NRPS NcpB is predicted to catalyze an NAD(P)H-mediated hydride transfer to the heptapeptidyl-S-enzyme intermediate NH(2)-Tyr-Gly-DGln-Ile-Ser-mPro-Leu/Phe-S-NRPS to yield a linear heptapeptide aldehyde that is subsequently captured intramolecularly with the amino group of the N-terminal amino acid residue tyrosine to form a stable imine bond. While a few C-terminal reductases associated with NRPSs have been identified, the ncp reductase is the first to mediate imine macrocyclization involving peptide N- and C-termini. Biochemical analysis of the NcpA1 and NcpB1 adenylation domains coupled with the recent characterization of the (2S,4S)-5-hydroxyleucine dehydrogenase NcpD, which is involved in the biosynthesis of the nonproteinogenic amino acid residue L-4-methylproline from L-leucine, support the involvement of this cluster in nostocyclopeptide biosynthesis.