Cytochrome P450 initiates degradation of cis-dichloroethene by Polaromonas sp. strain JS666

Abstract

Polaromonas sp. strain JS666 grows on cis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway for cDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step of cDCE degradation. Both the transient accumulation of dichloroacetaldehyde in cDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation of cDCE through dichloroacetaldehyde. The mechanism minimizes the formation of cDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that the cDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes.

Fig 1

Map of region cloned into pET21a. Key: F, ferredoxin gene; Red, reductase gene; and P450, cytochrome P450 gene. Clones also contain the native ribosome binding site (RBS) and 562 bp of intergenic DNA. Gene loci are indicated above the genes.

Fig 2

Potential initial steps for cDCE degradation in JS666 include reduction (I), hydration (II), glutathione addition (III), monooxygenation to an aldehyde (IV), monooxygenation to an epoxide (V), or reductive dehalogenation (VI). GSH, reduced glutathione; SG, glutathione conjugated to the organic compound, where the H (hydrogen) has been replaced by the organic compound.

Fig 3

Dependence of cDCE and DCA degradation by JS666 on oxygen. (A) cDCE-grown cells were added to N₂-sparged (□) and unsparged (•) serum bottles. Reactions were initiated by addition of cDCE to the bottles. Arrow, addition of 5 ml of air. (B) cDCE and DCA were added as neat solutions to N₂-sparged serum bottles. Reactions were initiated by adding 1 ml of cDCE-grown sparged cells to bottles. Arrow, addition of 1 ml of O₂. Error bars indicate 1 standard deviation.

Fig 4

Effect of cytochrome P450 inhibitors on JS666. (A) Time course of cDCE degradation with no inhibitor (•), 200 μM metyrapone (○), or 200 μM phenylhydrazine (□). (B) Concentration-dependent effect of metyrapone on cDCE (•) and DCA (■) degradation by JS666. cDCE and DCA were provided at 100 μM.

Fig 5

Expression of cytochrome P450. Lane 1, uninduced clone; lane 2, induced clone expressing Bpro_5301 (47.49 kDa), Bpro_5300 (43.18 kDa), and Bpro_5299 (11.29 kDa); lane 3, clone expressing Bpro_5301 with a 6× His tag (48.32 kDa); lane 4, clone expressing Bpro_5299 with a 6× His tag (12.12 kDa); lane 5, clone expressing LacZ (120 kDa).

Fig 6

(A) Degradation of cDCE by JS666 (solid lines and symbols; OD₆₀₀ ≈ 1) and transformation by JS595 (dashed lines, open symbols; OD₆₀₀ ≈ 40) and uninoculated control (open circle, dotted line). Circles, cDCE; squares, 2,2-dichloroacetaldehyde; diamonds, unknown metabolite. (B) Mass spectrum of unknown. (C) Mass spectrum of 2, 2-dichloroacetaldehyde.

Fig 7

Biotransformation of cDCE in cell extracts of JS595 expressing cDCE monooxygenase. Complete mixtures contained cDCE, phosphate buffer, cell extract, and NADH. Controls lacked either NADH or cell extract. Results are for cDCE in complete mixtures (•), cDCE in controls (■), final dichloroacetaldehyde in complete mixtures (○), and final dichloroacetaldehyde in controls (□). cDCE was allowed to equilibrate for 5 min, and then reactions were initiated by adding NADH.

Fig 8

Proposed cDCE and DCA degradation pathways. Solid arrows, major pathways supported by the results of this study; dashed arrows, potential minor pathways.