Anthramycin is a potent antitumor and antibiotic benzodiazepine alkaloid produced by the thermophilic actinomycete Streptomyces refuineus.  We have recently identified and cloned the nonribosomal peptide biosynthetic cluster of anthramycin and have developed several possible mechanisms for anthramycin biosynthesis.  Chemical complementation studies of gene knockouts provide evidence methylation occurs after biosynthesis of the intermediate 3-hydroxyanthranilic acid, a known primary metabolite.  The anthramycin cluster contains two NRPS genes, one of which, Orf21, shows low sequence similarity to known A-domains including actinomycin synthetase, which has previously been demonstrated to activate 4-methyl-3-hydroxyanthranilic acid.  We have heterologously expressed orf21 and established the A-domain specificity using a novel mass spectrometry based method.