Characterization of the N-methyltransferases involved in the biosynthesis of toxoflavin, fervenulin and reumycin from Streptomyces hiroshimensis ATCC53615Electronic supplementary information (ESI) available: Spectroscopic data, as well as the NMR and MS spectra of 1-3 and 9-10. See DOI: 10.1039/c8ob02847h

Toxoflavin ( 1 ), fervenulin ( 2 ), and reumycin ( 3 ), known to be produced by plant pathogen Burkholderia glumae BGR1, are structurally related 7-azapteridine antibiotics. Previous biosynthetic studies revealed that N -methyltransferase ToxA from B. glumae BGR1 catalyzed the sequential methylation at N6 and N1 in pyrimido[5,4- e ]-as-triazine-5,7(6 H ,8 H )-dione ( 4 ) to generate 1 . However, the N8 methylation of 4 in the biosynthesis of fervenulin remains unclear. To explore the N -methyltransferases required for the biosynthesis of 1 and 2 , we identified and characterized the fervenulin and toxoflavin biosynthetic gene clusters in S. hiroshimensis ATCC53615. On the basis of the structures of intermediates accumulated from the four N -methyltransferase gene inactivation mutants and systematic enzymatic methylation reactions, the tailoring steps for the methylation order in the biosynthesis of 1 and 2 were proposed. The N -methylation order and routes for the biosynthesis of fervenulin and toxoflavin in S. hiroshimensis are more complex and represent an obvious departure from those in B. glumae BGR1. Four SAM-dependent N -methyltransferases are involved in the biosynthesis of toxoflavin, fervenulin, and reumycin from Streptomyces hiroshimensis ATCC53615.