title: Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair.
abstract: Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.
authors.affiliation: Center for Oceans and Human Health, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Abrahim
authors.initials: A
authors.lastname: El Gamal
authors.affiliation: Center for Oceans and Human Health, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Vinayak
authors.initials: V
authors.lastname: Agarwal
authors.affiliation: Center for Oceans and Human Health, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Stefan
authors.initials: S
authors.lastname: Diethelm
authors.affiliation: Center for Oceans and Human Health, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Imran
authors.initials: I
authors.lastname: Rahman
authors.affiliation: Center for Oceans and Human Health, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Schorn
authors.affiliation: Smithsonian Marine Station at Fort Pierce, Fort Pierce, FL 34949;
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Sneed
authors.affiliation: Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037; Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, CA 92037;
authors.forename: Gordon V
authors.initials: GV
authors.lastname: Louie
authors.affiliation: Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02543;
authors.forename: Kristen E
authors.initials: KE
authors.lastname: Whalen
authors.affiliation: Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02543;
authors.forename: Tracy J
authors.initials: TJ
authors.lastname: Mincer
authors.affiliation: Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037; Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, CA 92037;
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
authors.affiliation: Smithsonian Marine Station at Fort Pierce, Fort Pierce, FL 34949;
authors.forename: Valerie J
authors.initials: VJ
authors.lastname: Paul
authors.affiliation: Center for Oceans and Human Health, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093 bsmoore@ucsd.edu.
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Moore
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000378:metabolism; D059001:Aquatic Organisms / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001966:Bromine / Q000737:chemistry; D018360:Crystallography, X-Ray; D005415:Flavins / Q000378:metabolism*; D054879:Halogenation / Q000502:physiology*; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D011758:Pyrroles / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D011758:Pyrroles; D001966:Bromine
keywords: biosynthesis; enzymology; halogenation; natural products