The overall aim of the proposed research is to understand the molecular mechanisms controlling the biosynthesis of and cellular resistance to the antitumor antibiotic, mitomycin C. This important chemotherapeutic agent is biosynthetically derived from a shikimate pathway metabolite (3-amino-5-hydroxybenzoic acid) and D-glucosamine. In this work, molecular genetic, biochemical and chemical approaches will be used to obtain information on the functional role of the set of genes and enzymes involved in constructing this important anticancer drug. Our initial work demonstrated that Streptomyces lavendulae (the mitomycin producer) had at least two genetic loci (mcr and mrd) that specify resistance to mitomycin. Identification of cosmid clones containing DNA adjacent to the resistance genes revealed that mitomycin biosynthetic genes are clustered around the mitomycin resistance determinant (mrd). Using probes for shikimate pathway genes, homologs to the dehydroquinase, dehydroquinate synthase, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and 3-amino-5-hydroxybenzoic acid synthase (AHBAS) were identified among a total of 47 genes within the 55 kb cluster. With this information in hand, gene disruption/replacement, mutant complementation, and biochemical experiments will be performed to probe the precise function of the individual mitomycin biosynthetic enzymes. This information will be used to identify and characterize the mechanism and specificity of the enzyme(s) responsible for coupling the AHBA precursor to the D-glucosamine sugar moiety. Subsequently, studies will be initiated to understand and manipulate enzymes involved in establishing the core mitosane structure and the specificity of tailoring enzymes that provide molecular diversity to this significant class of metabolites. Concurrently, our work will continue on the resistance mechanisms that provide cellular self-protection against mitomycins in the producing organism. Overall, this work will provide an important theoretical and experimental base for future combinatorial biology-based production of novel AHBA-derived natural products using molecular genetic technology.
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