Incorporation of halogen atoms into drug molecules often increases biological activity as is the case for the potent proteasome inhibitor salinosporamide A, a chlorinated natural product from the marine actinomycete Salinispora tropica currently in development for multiple myeloma treatment. Common enzymatic strategies for C-Cl bond formation involve oxidation of chloride ion to form reactive electrophilic or radical species which then function as halogenating agents. The chlorinase SalL involved in salinosporamide A biosynthesis however uses ground state chloride to displace L-methionine from S-adenosyl-L-methionine (SAM) and form 5’-chloro-5’deoxyadenosine (5’-ClDA) in a rarely observed nucleophilic substitution route analogous to fluorinase of Streptomyces cattleya, a fluoroacetate producer. Recombinant SalL protein accepts also bromide and iodide as substrates but not fluoride. Crystal structures of SalL wild-type and active site mutants bound with substrates or products confirm the S_N2 type mechanism and illuminate halide specificity in this newly characterized halogenase family.

Remarkably, the discovery of common committed steps for the biosynthesis of a chlorinated and a fluorinated natural product opens the door for rationally engineering new drug candidates through biotechnology. Hence, administration of synthetic 5’-FDA to a salL⁻ mutant strain led to the production of fluorosalinosporamide.

Moreover, SalL and fluorinase belong to a family of over one hundred archaeal and bacterial proteins with unknown function (DUF62). Based on sequence alignments, our structure–function studies in SalL and biochemical data on a DUF62 member, these relatives are unlikely to be SAM-dependent halogenases but could help understand their evolution.