Multiple amide-bond formation in streptothricin biosynthesis catalyzed by a stand-alone adenylation domain

Streptothricins (STs; syn. nourseothricins, racemomycins, and yazumycins), which inhibit protein biosynthesis in prokaryotic cells, and strongly inhibit the growth of eukaryotes, are a group of N-glycoside toxic antibiotics. The first member of this group, ST-F, was isolated by Waksman from Streptomyces lavendulae in 1943. All ST-related compounds consist of a carbamoylated D-gulosamine to which the (3S)-3,6-diaminohexanoic acid (l-ß-lysine) oligopeptide (one to seven residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached.

Here we report on three unusual non-ribosomal peptide synthetases involved in the biosynthesis of l-ß-lysine oligopeptides: open reading frame (ORF) 5 (stand-alone adenylation (A)-domain); ORF 18 (thiolation (T)- and condensation (C)-domains); and ORF 19 (stand-alone A-domain). Biosynthesis was initiated by adenylation of l-b-lysine in ORF 5. The resulting l-b-lysyl-O-AMP was exclusively loaded on the T-domain of ORF 18. l-ß-lysine molecules adenylated by ORF 19 were not loaded, but instead were directly used as extending units for the elongation of an l-ß-lysine peptide chain on ORF 18. Surprisingly, amide-bond formations for the elongation were iteratively catalyzed by ORF 19 itself. The C-domain of ORF 18 catalyzed the condensation reaction between the l-ß-lysine oligopeptides covalently bound to ORF 18 and a freely-diffusible l-ß-lysine (or an ST intermediate), thereby releasing l-ß-lysine oligopeptides.