Biosynthesis of polybrominated aromatic organic compounds by marine bacteria

Abstract

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention because of their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominases revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds.

Figure 1. Structures and sources of marine polybrominated natural products

(a) Representative structures of polybrominated molecules produced anthropogenically (BDE-47), or microbially (1–3), and their derivatives detected in the environment. (b) Total ion chromatogram (TIC) (black curve) from LC-MS/MS analysis of an organic extract of P. luteoviolacea 2ta16 demonstrating production of the 1–3. Peaks labeled ● and ▲ were later correlated to OH-BDEs 11 and 12, respectively (vide infra). (c) Organization of bmp gene loci in marine bacteria. Note that M. mediterranea MMB-1 encodes a putative permease between bmp3 and bmp4.

Figure 2. Genetic basis of the Bmp pathway

(a) Total ion chromatograms (TICs) for organic extracts of E. coli expressing the P. luteoviolacea 2ta16 bmp gene cluster and selected gene deletions. Molecule 2 is only present in high abundance in the absence of bmp5 as E. coli expressing both bmp5 and bmp7 preferentially produce 1 and other coupled bromophenols. Retention times of 5 and 7 are nearly identical; hence, for the sake of clarity, the more abundant species is highlighted in chromatograms where both are present (i.e., bmp1–8, and Δbmp7). In addition to 1–7, the bmp1–8 extract contains other polybrominated phenol species. Peaks labeled ● and ▲ were later correlated to OH-BDEs 11 and 12 (vide infra). TIC for an organic extract of E. coli expressing bmp1–8 cultured in the absence of bromide is shown as a negative control. (b) Chemical structures of bromophenol and bromopyrrole monomers 4–7.

Figure 3. In vitro reconstitution of activity for brominase Bmp2

Assay for the activity of Bmp2 relies on the detection of MS1 ions for tryptic peptide fragments of Bmp1 corresponding to (a) holo-S-Bmp1, (b) pyrrolyl-S-Bmp1, (c) monobromopyrrolyl-S-Bmp1 (d) dibromopyrrolyl-S-Bmp1 and (e) tribromopyrrolyl-S-Bmp1. All MS1 ions (a–e, shaded in yellow) bear a +7 charge, as identified by their isotopic distributions. MS/MS fragmentation of the shaded MS1 ions generates distinct diagnostic MS2 ions that are shown by their corresponding chemical structures. Note that the dehydrated cyclopantetheinyl MS2 ion for panel a is abbreviated for clarity in panels b–e. Upon transfer of pyrrolyl-S-CoA (16) to apo-S-Bmp1(ACP) by the B. subtilis phosphopantetheinyl transferase Sfp (described in the Online Methods section), pyrrolyl-S-Bmp1 is generated, that is mono-, di- and tribrominated by Bmp2 in the presence of NADPH, KBr and E. coli flavin-reductase SsuE. Calculation for MS1 peptide masses and peptide identification protocol, as inspired by prior reports^, is described in detail in the Supplementary Note 1. Note that the expression of Bmp1 in E. coli generates a mixture of apo-Bmp1 and holo-S-Bmp1. Of these, only apo-Bmp1 participates in the Bmp2 assay due to its amenability for acylation catalyzed by the Sfp transferase. Holo-S-Bmp1 remains unmodified as shown in panel a.

Figure 4. Biosynthesis of bromophenols by flavin-dependent decarboxylase-brominase Bmp5

(a) Bmp5 was incubated with 4-HBA, KBr, NADPH, and FAD at 30 °C in 20 mM Tris-HCl (pH 8.0) buffer. At indicated time points, 40 µL assay volume was withdrawn and quenched by the addition of 16 µL MeCN + 0.35% TFA. HPLC separation of the substrate, intermediate and products of the Bmp5 reaction are shown as black traces after the following reaction times: 1 min, 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 360 min, 540 min and 1440 min. HPLC traces corresponding to synthetic standards analyzed under identical chromatographic conditions are shown in red. (b) Time-dependent conversion of 8 to 4, and of 9 to 5 was monitored in an identical fashion, and the area under the substrate peaks was integrated and plotted against reaction time to compare the relative rates for the consumption of 4-HBA, 8 and 9 by Bmp5. Experiments were conducted in triplicate; data represent mean values ± s.d. (c) A proposed reaction scheme for the conversion of 4-HBA to 4 catalyzed by Bmp5.

Figure 5. Enzymatic synthesis of polybrominated biphenyls and OH-BDEs

(a) CYP450 Bmp7 dimerizes two molecules of 4 to generate biphenyls 1 and 10, and OH-BDEs 11–13 (red curve). The curve in black represents the negative control in the absence of the enzyme. (b) Chemical structures of polybrominated biphenyls and OH-BDEs generated by the bmp pathway as identified in this study. Coupling bonds generated by Bmp7 are shown in red. Also shown is 2’-MeO-BDE-68, the methoxylated derivative of 13.

Figure 6. Bi-modular scheme for the biosynthesis of polybrominated marine natural products by the bmp pathway

(a) Chorismate, the precursor for bromophenols, is converted to 4-HBA by chorismate lyase (CL) Bmp6, and then to 4–5 by flavin-dependent halogenase (Hal) Bmp5. The CYP450 coupling (C) enzyme Bmp7 generates a suite of diverse polybrominated biphenyls (1 and 10) and OH-BDEs (11–14) from 4 and 5. The electron transfer partners for Bmp7, Bmp9 and Bmp10, are omitted for clarity. The bromopyrroles are derived from L-proline. Acylation of L-proline to the ACP domain of Bmp1 by the proline adenyltransferase (A) Bmp4 initiates its oxidation by the flavin-dependent dehydrogenase (DH) Bmp3 and tribromination by the flavin-dependent halogenase (Hal) Bmp2. The TE domain of Bmp1 likely catalyzes the offloading of a carboxylic acid intermediate that is decarboxylated (D) by carboxymuconolactone decarboxylase homolog Bmp8 to 6. 6 can be dimerized by Bmp7 to generate 2, or with 4 to generate heterodimers such as 3 and 15. During the homodimerization of 6, we also observed the formation of 7. The coloring scheme is consistent with Fig. 1c. (b) Proposed steps for radical generation (i), rearragement (ii–iv) and coupling of 4 by Bmp7 to generate biphenyls and OH-BDEs.