Genetic localization and molecular characterization of two key genes (mitAB) required for biosynthesis of the antitumor antibiotic mitomycin C

Mitomycin C (MC) is an antitumor antibiotic derived biosynthetically from 3 -amino-5-hydroxybenzoic acid (AHBA), D glucosamine, and carbamoyl phosphate . A gene (mitA) involved in synthesis of AHBA has been identified and found to be linked to the MC resistance locus, mrd, in Streptomyces lavendulae. Nucleotide sequence analysis showed that mitA encodes a 388-amino-acid prot ein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediterranei, as well as,vith two additional AHBA synth ases from related ansamycin antibiotic-producing microorganisms. Gene disru ption and site-directed mutagenesis of the S. lavendulae chromosomal copy o f mitA completely blocked the production of MC. The function of mitA was co nfirmed by complementation of an S. lavendulae strain containing a K191A mu tation in MitA with AHBA. A second gene (mitB) encoding a 272-amino-acid pr otein (related to a group of glycosyltransferases) was identified immediate ly downstream of mitA that upon disruption resulted in abrogation of MC syn thesis. This work has localized a cluster of key genes that mediate assembl y of the unique mitosane class of natural products.