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Discovery and characterization of a marine bacterial SAM-dependent chlorinase

Nature Chemical Biology volume 4, pages 69–74 (2008)Cite this article

Abstract

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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Figure 1: Biosynthesis and structures of salinosporamides A and B.
Figure 2: Structures of wild-type SalL and the Y70T mutant.
Figure 3: SalL double-mutant Y70T G131S and structural comparison with fluorinase.

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Acknowledgements

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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Author notes

  1. Alessandra S Eustáquio and Florence Pojer: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA

    Alessandra S Eustáquio & Bradley S Moore

  2. Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, 92037, California, USA

    Florence Pojer & Joseph P Noel

  3. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA

    Bradley S Moore

Authors
  1. Alessandra S Eustáquio
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  2. Florence Pojer
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  3. Joseph P Noel
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  4. Bradley S Moore
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Contributions

A.S.E. and F.P. contributed equally to this paper. A.S.E. performed the genetic and biochemical experiments, F.P. and A.S.E. crystallized SalL, F.P. determined the structure and performed the sedimentation velocity studies. All authors discussed the results and wrote and commented on the manuscript.

Corresponding author

Correspondence to Bradley S Moore.

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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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