Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation

Abstract

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides1. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle2,3. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria4,5, with substrates including polychlorinated biphenyls or dioxins6,7. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies8,9,10,11,12. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon–cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies13, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen–cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Characterization of NpRdhA as an ortho-dibromophenol reductase.
Figure 2: Crystal structure of NpRdhA in the resting state.
Figure 3: EPR spectroscopy of NpRdhA reveals a direct halogen–cobalamin interaction.
Figure 4: NpRdhA proposed mechanism.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 4RAS.

References

  1. 1

    Stringer, R. & Johnston, P. Chlorine and the Environment. An Overview of the Chlorine Industry (Kluwer Academic, 2001)

    Google Scholar 

  2. 2

    Öberg, G. The natural chlorine cycle–fitting the scattered pieces. Appl. Microbiol. Biotechnol. 58, 565–581 (2002)

    Article  Google Scholar 

  3. 3

    Gribble, G. W. Occurrence of halogenated alkaloids. Alkaloids Chem. Biol. 71, 1–165 (2012)

    CAS  Article  Google Scholar 

  4. 4

    Smidt, H. & de Vos, W. M. Anaerobic microbial dehalogenation. Annu. Rev. Microbiol. 58, 43–73 (2004)

    CAS  Article  Google Scholar 

  5. 5

    Leys, D., Adrian, L. & Smidt, H. Organohalide respiration: microbes breathing chlorinated molecules. Phil. Trans. R. Soc. Lond. B 368, 20120316 (2013)

    Article  Google Scholar 

  6. 6

    Adrian, L., Szewzyk, U., Wecke, J. & Görisch, H. Bacterial dehalorespiration with chlorinated benzenes. Nature 408, 580–583 (2000)

    CAS  ADS  Article  Google Scholar 

  7. 7

    Bunge, M. et al. Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium. Nature 421, 357–360 (2003)

    CAS  ADS  Article  Google Scholar 

  8. 8

    Wohlfarth, G. & Diekert, G. Anaerobic dehalogenases. Curr. Opin. Biotechnol. 8, 290–295 (1997)

    CAS  Article  Google Scholar 

  9. 9

    van de Pas, B. A. et al. Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans. J. Biol. Chem. 274, 20287–20292 (1999)

    CAS  Article  Google Scholar 

  10. 10

    Krasotkina, J., Walters, T., Maruya, K. A. & Ragsdale, S. W. Characterization of the B12- and iron-sulfur-containing reductive dehalogenase from Desulfitobacterium chlororespirans. J. Biol. Chem. 276, 40991–40997 (2001)

    CAS  Article  Google Scholar 

  11. 11

    Neumann, A. et al. Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans: substrate specificity of the native enzyme and its corrinoid cofactor. Arch. Microbiol. 177, 420–426 (2002)

    CAS  Article  Google Scholar 

  12. 12

    Maillard, J. et al. Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus. Appl. Environ. Microbiol. 69, 4628–4638 (2003)

    CAS  Article  Google Scholar 

  13. 13

    Banerjee, R. & Ragsdale, S. W. The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes. Annu. Rev. Biochem. 72, 209–247 (2003)

    CAS  Article  Google Scholar 

  14. 14

    Brown, K. L. Chemistry and enzymology of vitamin B12. Chem. Rev. 105, 2075–2150 (2005)

    CAS  Article  Google Scholar 

  15. 15

    Chen, K. et al. Molecular characterization of the enzymes involved in the degradation of a brominated aromatic herbicide. Mol. Microbiol. 89, 1121–1139 (2013)

    CAS  Article  Google Scholar 

  16. 16

    Koutmos, M., Gherasim, C., Smith, J. L. & Banerjee, R. Structural basis of multifunctionality in a vitamin B12-processing enzyme. J. Biol. Chem. 286, 29780–29787 (2011)

    CAS  Article  Google Scholar 

  17. 17

    Stroupe, M. E. & Getzoff, E. D. In Tetrapyrroles: Their Birth, Life, and Death (eds Warren, M. J. & Smith, A. ) 373–387 (Landes Bioscience, 2005)

    Google Scholar 

  18. 18

    Van Doorslaer, S. et al. Axial solvent coordination in “base-off” cob(II)alamin and related co(II)-corrinates revealed by 2D-EPR. J. Am. Chem. Soc. 125, 5915–5927 (2003)

    CAS  Article  Google Scholar 

  19. 19

    Bayston, J. H., Looney, F. H., Pilbrow, J. R. & Winfield, M. E. Electron paramagnetic resonance studies of cob(II)alamin and cob(II)inamides. Biochemistry 9, 2164–2172 (1970)

    CAS  Article  Google Scholar 

  20. 20

    de Jong, R. M. & Dijkstra, B. W. Structure and mechanism of bacterial dehalogenases: different ways to cleave a carbon-halogen bond. Curr. Opin. Struct. Biol. 13, 722–730 (2003)

    CAS  Article  Google Scholar 

  21. 21

    Grostern, A. & Edwards, E. A. Characterization of a Dehalobacter coculture that dechlorinates 1,2-dichloroethane to ethene and identification of the putative reductive dehalogenase gene. Appl. Environ. Microbiol. 75, 2684–2693 (2009)

    CAS  Article  Google Scholar 

  22. 22

    Miles, Z. D., McCarty, R. M., Molnar, G. & Bandarian, V. Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification. Proc. Natl Acad. Sci. USA 108, 7368–7372 (2011)

    CAS  ADS  Article  Google Scholar 

  23. 23

    Bommer, M. et al. Structural basis for organohalide respiration. Science http://dx.doi.org/10.1126/science.1258118 (2 October 2014)

  24. 24

    Lai, Q., Li, G. & Shao, Z. Genome sequence of Nitratireductor pacificus type strain pht-3B. J. Bacteriol. 194, 6958 (2012)

    CAS  Article  Google Scholar 

  25. 25

    Gamer, M. et al. A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium. Appl. Microbiol. Biotechnol. 82, 1195–1203 (2009)

    CAS  Article  Google Scholar 

  26. 26

    Moore, S. J. et al. Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12). Proc. Natl Acad. Sci. USA 110, 14906–14911 (2013)

    CAS  ADS  Article  Google Scholar 

  27. 27

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    CAS  Article  Google Scholar 

  28. 28

    Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)

    CAS  Article  Google Scholar 

  29. 29

    Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010)

    CAS  Article  Google Scholar 

  30. 30

    Langer, G. et al. Automated molecular model building for X-ray crystallography using ARP/Warp version 7. Nature Protocols 3, 1171–1179 (2008)

    CAS  Article  Google Scholar 

  31. 31

    Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Frisch, M. J. et al. Gaussian 09 (Gaussian, revision B.01, 2010)

  33. 33

    Morris, G. M. et al. Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009)

    CAS  Article  Google Scholar 

  34. 34

    Liu, H., Kornobis, K., Lodowski, P., Jaworska, M. & Kozlowski, P. M. TD-DFT insight into photodissociation of the Co-C bond in coenzyme B12. Front. Chem 1, 1–12 (2013)

    CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by an ERC grant (DEHALORES206080) to D.L. S.H. is a BBSRC David Phillips research fellow. C.P.Q. is supported by CONICYT Chile. We thank Diamond Light Source for access to beamline IO4 (proposal number MX8997) that contributed to the results presented here; R. Biedendieck (Technische Universität Braunschweig, Germany), S. Moore and M. Warren (University of Kent, UK) for providing plasmids and assistance with B. megaterium molecular biology; the assistance given by IT Services and the use of the Computational Shared Facility at The University of Manchester; and A. Munro (University of Manchester) for critical comments and discussion.

Author information

Affiliations

Authors

Contributions

K.A.P.P., M.S.D., K.F. and H.S. were involved in rdhA heterologous production screening. K.A.P.P. obtained heterologous expression of active NpRdhA in B. megaterium. C.P.Q. crystallized and solved the NpRdhA structure with assistance from D.L. and C.L.; C.P.Q., K.A.P.P., M.S.D. and K.F. performed kinetic studies; K.A.P.P. and F.A.C. prepared and analysed NpRdhA variants. K.F. and S.E.J.R. performed and analysed EPR experiments. S.H. performed docking and DFT calculations. All authors discussed the results and participated in writing the manuscript. D.L. initiated and directed this research.

Corresponding author

Correspondence to David Leys.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Phylogenetic tree of functionally characterized reductive dehalogenases (RdhAs).

Those in bold have been purified and characterized in vitro. Those in italics have been identified from crude lysate assays via native PAGE or knock out/knock in. Those in plain text have implied substrate range based on transcriptional regulation. The sequence accession codes are as follows: AAD44542, Desulfitobacterium dehalogenans ATCC 51507 CprA; P81594, Desulfitobacterium hafniense DCB-2 CprA1; AAQ54585, Desulfitobacterium hafniense PCP-1 CprA5; AAL84925, Desulfitobacterium chlororespirans CprA; WP_019849102, Desulfitobacterium sp. PCE-1; AAK06764, Desulfitobacterium hafniense PCP-1 CprA3; AAO60101, Desulfitobacterium hafniense PCE-S PceA; O68252, Dehalospirillum multivorans PceA; AAF73916, Dehalococcoides mccartyi TceA; YP_181066, Dehalococcoides mccartyi 195 PceA; AAW80323, Desulfitobacterium hafniense Y51 PceA; CAD28790, Dehalobacter restrictus PceA; CAD28792, Desulfitobacterium hafniense TCE1 PceA; YP_001214307, Dehalococcoides mccartyi BAV1 BvcA; YP_003330719, Dehalococcoides mccartyi VS VcrA; ACF24863, Dehalococcoides sp. MB MbrA; YP_307261, Dehalococcoides mccartyi CBDB1 CbrA; BAE45338, Desulfitobacterium sp. KBC1 PrdA; BAE45337, Desulfitobacterium sp. KBC1 CprA; ACH87594, Dehalobacter sp. WL DcaA; CAJ75430, Desulfitobacterium dichloroeliminans LMG P-21439 DcaA; AFQ20272, Dehalobacter sp. CF CfrA; AFQ20273, Dehalobacter sp. DCA DcrA; AAG46187, Desulfitobacterium sp. PCE1 CprA; YP_002457196, Desulfitobacterium hafniense DCB-2 RdhA3; YP_001475501, Shewanella sediminis HAW-EB3 PceA; WP_015585978, Comamonas sp. 7D-2 BhbA; WP_008597722, Nitratireductor pacificus NpRdhA.

Extended Data Figure 2 EPR spectroscopic analysis of reduced NpRdhA (79 μM).

a, b, X-band EPR spectra of NpRdhA as isolated, 150 μM (a), and reduced using EDTA, deazaflavin and light, 79 μM (b). c, X-band (9.39 GHz) continuous wave EPR spectrum of reduced NpRdhA recorded at 10 K showing a 2×[4Fe-4S]1+ signal indicative of two magnetically interacting 4Fe–4S clusters due to features marked with a dotted arrow; g values marked for rhombic [4Fe–4S]1+ signal. d, X-band continuous wave EPR spectrum of reduced NpRdhA at 40 K. e, X-band continuous wave EPR spectrum of reduced NpRdhA at 80 K showing small base-on cob(ii)alamin signal with g values marked. f, Subtraction of e from d reveals a second axial [4Fe–4S]1+ signal, with g values as marked, demonstrating that we can observe EPR signals from two different 4Fe–4S clusters within NpRdhA. Experimental parameters: microwave power 0.5 mW, field modulation frequency 100 KHz, field modulation amplitude 7 G, temperatures as given.

Extended Data Figure 3 Crystal structure of NpRdhA.

a, Stereo view of an overlay of the NpRdhA cobalamin-binding domain (in blue), the N-terminal non-functional cobalamin-binding domain (in light blue) and the human vitamin B12-processing enzyme CblC (in green). The cobalamin cofactor is bound in a similar base-off manner by both NpRdhA and CblC. The NpRdhA non-functional cobalamin-binding domain does contain an irregular water-filled cavity corresponding to the cofactor binding site, but no longer contains any of the residues implied in cofactor binding. b, Representation of the NpRdhA cofactors in space-filling spheres. The second [4Fe–4S] cluster is in van der Waals contact with the corrin ring; the B pyrrol d-amide corrin moiety is lined along one side of the cluster and forms a hydrogen bond to one of the S ions. c, Detailed view of the NpRdhA fifth ligand-binding site. Residues in close contact with the chloride ligand are shown in atom coloured sticks (colour coded as in Fig. 2). The 2Fo − Fc map is contoured at 1σ and shown as a blue mesh. The Tyr 426–Lys 488 distance is 2.7 Å. d, Model of the NpRdhA–substrate complex (colour coded as in Fig. 2e) with the FoFc omit map for an iodide-soaked crystal (to 3.5 Å; contoured at 6σ) depicted by a green mesh. Difference density can be clearly seen at positions corresponding to those predicted to accommodate the bromide atoms of the substrate. e, Putative structure of an organometallic substrate–cobalamin intermediate within the NpRdhA active site. The substrate was positioned to minimize close contacts with active site residues. Severe clashes can be observed between the substrate and the corrin ring (highlighted by red lines).

Extended Data Figure 4 Additional EPR spectroscopic data.

a, b, Identification of contaminating EPR signals in aerobically purified NpRdhA (150 μM). a, X-band continuous wave EPR spectrum showing a [3Fe–4S]1+ cluster signal isolated by subtraction of the spectrum recorded at 20 K from that recorded at 12 K. b, X-band continuous wave EPR spectrum showing cob(iii)alamin-O2•− (superoxide) signal isolated by subtracting the spectrum recorded at 20 K from that recorded at 30 K followed by subtraction of a proportion of the [3Fe–4S]1+ signal. Neither of these signals quantitates to more than 4% of the total EPR signal in the protein as isolated. Experimental parameters: microwave power 0.5 mW, field modulation frequency 100 KHz, field modulation amplitude 5 G, temperatures as given. c, d, Binding of 3,5-dichloro-4-hydroxybenzoate to 150 μM NpRdhA leads to a spectrum exhibiting multiple and splitting of the spectrum and no apparent chlorine superhyperfine coupling () (spectrum d). This suggests relatively disordered binding and possibly substrate movement within the active site on the nanosecond timescale even at the cryogenic temperatures employed in the EPR experiment. Such disorder and dynamics may explain the poor efficacy of this substrate relative to the dibromo analogue (see Fig. 1e) in addition to precluding analysis of the EPR spectrum.

Extended Data Figure 5 Structure of the NpRdhA active site DTF model.

a, b, Chemical structures used for DFT calculations (a) and overlay of the optimized active site models in the Co(ii) (pink carbons) and Co(i) (green carbons; Br is shown as a discrete sphere) oxidation states (b). The Co and the three atoms indicated with arrows (a) were fixed during optimization. Selected parameters are given in Extended Data Table 2 and Cartesian coordinates of the optimized structures are included in Supplementary Table 1. The Cbl–Br models comprise the ‘trimmed’ cobalamin with a single axial Br ligand and contain 91 atoms (40 heavy atoms). The full active site model contains 148 atoms (66 heavy atoms).

Extended Data Figure 6 Characterization of NpRdhA mutants.

a, UV–visible spectra of NpRdhA variants normalized using the A280 absorbance. Mutants Y426F (107 μM), K488Q (100 μM) and R552L (95 μM) have a similar profile as the wild-type enzyme (WT, 176 μM) purified from B. megaterium. NpRdhA wild type purified from E. coli (lacking the corrinoid cofactor; 250 μM) is shown for comparison. bd, Continuous wave X-band EPR at 30 K for NpRdhA mutants. b, R552L mutant (75 μM); c, K488Q mutant (65 μM); d, Y426F mutant (65 μM). Each shows the presence of cob(ii)alamin which is base on in R552L and base off in the other two mutants. Experimental parameters: microwave power 0.5 mW, modulation frequency 100 KHz, modulation amplitude 7 G.

Extended Data Figure 7 Mechanistic proposal for biological reductive dehalogenation.

Our proposed NpRdhA mechanism is fundamentally different from those for other B12-containing enzymes13,14, but also differs from the hydrolytic dehalogenases, which use an SN2 mechanism whereby either an activated water molecule or a catalytic Asp residue attacks the substrate carbon5. The hydrolytic dehalogenases contain a specific halogen-binding site that is believed to contribute to stabilization of the transition state and to facilitate departure of the halide leaving group. In contrast, we propose that the reductive dehalogenase uses the cobalamin cofactor to attack the substrate halogen atom itself, leading to C–halogen bond breakage concomitant with protonation of the leaving group. Distinct variations on this theme could occur within the RdhA family: aliphatic organohalide reductases could operate via heterolytic C–halogen bond cleavage concomitant with halogen–Co(iii) bond formation and substrate protonation (a). Those acting on (unactivated) aromatic organohalides are likely to operate via a radical substrate intermediate using homolytic C–halogen bond cleavage (b), while certain reductive dehalogenases have been shown to catalyse vicinal reduction or dihalo-elimination, and we propose that formation of the Co–halogen bond occurs concomitant with leaving of the vicinal halogen atom (c).

Extended Data Table 1 Data collection, phasing and refinement statistics
Extended Data Table 2 EPR parameters for the spectra shown in Fig. 3
Extended Data Table 3 Selected parameters obtained from DFT calculations of model compounds

Supplementary information

Supplementary Table 1

This file contains the B12 DFT coordinates. (PDF 233 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Payne, K., Quezada, C., Fisher, K. et al. Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation. Nature 517, 513–516 (2015). https://doi.org/10.1038/nature13901

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing