Halogen atom incorporation into a scaffold of bioactive compounds often amplifies biological activity, as is the case for the anticancer agent salinosporamide A (1), a chlorinated natural product from the marine bacterium Salinispora tropica. Significant effort in understanding enzymatic chlorination shows that oxidative routes predominate to form reactive electrophilic or radical chlorine species. Here we report the genetic, biochemical and structural characterization of the chlorinase SalL, which halogenates S-adenosyl-L-methionine (2) with chloride to generate 5′-chloro-5′-deoxyadenosine (3) and L-methionine (4) in a rarely observed nucleophilic substitution strategy analogous to that of Streptomyces cattleya fluorinase. Further metabolic tailoring produces a halogenated polyketide synthase substrate specific for salinosporamide A biosynthesis. SalL also accepts bromide and iodide as substrates, but not fluoride. High-resolution crystal structures of SalL and active site mutants complexed with substrates and products support the SN2 nucleophilic substitution mechanism and further illuminate halide specificity in this newly discovered halogenase family.
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We kindly thank D. O'Hagan (University of St. Andrews) for providing 5′-FDA and 5′-BrDA standards, B. Gust and Plant Bioscience Limited for the REDIRECT technology kit for PCR targeting, A. Lapidus (Joint Genome Institute) for fosmid BHXS3930 and R. McGlinchey for valuable discussions. A.S.E. is a Tularik postdoctoral fellow of the Life Sciences Research Foundation, and F.P. is a Deutsche Forschungsgemeinschaft postdoctoral fellow. This work was supported by grants from the US National Oceanic and Atmospheric Administration (NA05NOS4781249 to B.S.M.), the US National Institutes of Health (CA127622 to B.S.M.) and the US National Science Foundation (MCB-023602 to J.P.N.). J.P.N. is an investigator of the Howard Hughes Medical Institute.
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Eustáquio, A., Pojer, F., Noel, J. et al. Discovery and characterization of a marine bacterial SAM-dependent chlorinase. Nat Chem Biol 4, 69–74 (2008). https://doi.org/10.1038/nchembio.2007.56
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