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Ring hydroxylating dioxygenases

Published on 24 January 2019

Dr. Yves Jouanneau
Laboratoire Chimie et Biologie des Métaux
17 avenue des Martyrs
38 054 Grenoble Cedex 09
Phone: 04 38 78 43 10
Fax: 04 38 78 54 87
Ring-hydroxylating dioxygenases (RHDs) form a large family of metalloenzymes that catalyze the incorporation of the two atoms of molecular oxygen into a wide range of carbon substrates. RHDs found in some bacteria can attack inherently very stable aromatic or polyaromatic molecules.
The interest in these enzymes is twofold:
• the reactions they catalyze are regio- and enantioselective, generating chiral molecules useful for synthetic chemistry,
• they catalyze the first step in the biodegradation of many toxic pollutants, sometimes persistent in the environment, such as polycyclic aromatic hydrocarbons (PAHs) (Jouanneau et al., 2011).

Structure and selectivity of dioxygenases

We have studied RHDs from bacterial isolates identified as members of the Mycobacterium or Sphingomonas genera. Remarkably, these enzymes are able to attack high molecular weight PAHs consisting of four aromatic rings. For example, the RHD from Sphingomonas CHY-1 is exceptional in its ability to oxidize 9 of 16 PAHs considered as priority pollutants by environmental agencies. This is the first characterized enzyme with a so broad substrate specificity, including the ability to oxidize a highly toxic 5-ring PAH, benzo[a]pyrene (Jouanneau et al., 2006).

Functional diagram of ring-hydroxylating dioxygenases.
The enzyme complex consists of three components. Two electrons from the oxidation of NADH are transferred via a reductase and a ferredoxin to an oxygenase component which catalyzes the stereoselective dihydroxylation of the substrate to form a

In collaboration with colleagues at the Brookhaven National Laboratory (Upton, NY, USA), the oxygenase component of the CHY-1 RHD has been crystallized and its 3D structure has been elucidated (Jakoncic et al., 2007a, 2007b). It is a hexamer α3ß3, where the active site is an atom of ferrous iron carried by each alpha subunit. This iron atom is located at the bottom of a deep hydrophobic pocket in which the substrate is positioned so as to allow the selective hydroxylation of two adjacent carbon atoms on one ring of the substrate.
In addition, through collaboration with a Belgian team, we have studied the catalytic activity of other RHDs, including that of Sphingomonas LB126 which has the particularity to catalyze angular dioxygenation of substrates like fluorene or dibenzofuran
(Schuler et al., 2008, 2009). The objective of this work is to better understand what determines at the molecular level, the catalytic selectivity of this kind of enzymes.

Crystal structure of the catalytic component of the RHD from Sphingomonas CHY 1.
3D representation of the structure of the hexamer α3ß3 (left) and the catalytic site in which one molecule of benzo [
a] pyrene has been modeled (right).

Biodiversity of soil bacteria and relevant enzymes involved in PAH degradation

Another aspect of our work aims to better understand the PAH biodegradation potential present in polluted soils. This project is based on the observation that only 5% of soil bacteria can be isolated and grown in the laboratory, and therefore most of the microbial biodiversity and biocatalytic richness of soil is unknown. The approach is based on a technique of in situ metabolic labeling with a stable isotope, known as stable isotope probing (SIP). This technique comprises the following steps: incubation of soil samples with a 13C-labeled substrate, namely phenanthrene, then extraction of the labeled DNA from soil, and finally identification of the bacteria capable of degrading PAHs based on biomarker analysis (16S rRNA).
In this way, the bacterial diversity of a soil from a treatment facility collecting hydrocarbon-contamnated road runoffs from a highway has been studied. Results showed that Betaproteobacteria represented by several poorly known bacterial genera were the major players in the biodegradation of PAHs in situ
(Martin et al., 2012). More recently, our work has focused on soil bacteria capable of degrading PAHs adsorbed on a hydrophobic sorbent. The analysis showed that the species of interest, growing in biofilm at the interface with the hydrophobic sorbent, were different from those previously identified in the same soil. Also, the nature of the bacterial genera that degrade PAHs depends on the geographical origin of the soil. Nevertheless, the main microorganisms capable of degrading PAHs in a tropical soil from Cameroon have been identified as species of the Rhodocyclaceae family, very closely related to those found in the soil under temperate climate (Kom Regonne et al., 2013).

Prevalence of Betaproteobacteria among soil bacteria capable of degrading PAHs.
The analysis involves 427 16S ribosomal RNA gene sequences amplified by PCR from 13C-labeled metagenomic DNA (SIP labeling for 5 days). The assignment of sequences to bacterial phyla or classes was made through the RDP Database. Proteobacteria from the Alpha (4%), Beta (67%)and Gamma (11%) classes were the most represented among soil bacteria able to degrade PAHs.

The SIP technique is also used to identify the enzymes involved in PAH degradation, including dioxygenases, by analyzing specific gene sequences from soil. Using partial RHD sequences retrieved from soil DNA, a novel method based on the construction of hybrid genes has been implemented to restore functional RHDs and measure their PAH oxidation activity. This approach is limited by the stability of hybrid proteins studied. However, analysis of more than one hundred RHD sequences showed that they could be classified into 5 distinct groups, of which only two showed significant homology with known enzymes (Martin et al., 2013)

To learn more about the richness of the soil bacterial biocatalysts associated with biodegradation, a metagenomic analysis was undertaken in collaboration with the Genoscope at Evry (France). A scaled-up SIP experiment was performed and the labeled DNA extracted from soil was subjected to high-throughput sequencing. Of a total of 283 Mb of metagenomic DNA deciphered, the greatest part (56%) was from Betaproteobacteria in agreement with our previous genotype analysis. Dozens of genes potentially encoding dioxygenases have been identified, most of them having little similarities with known enzymes. The structural genes of four RHDs have been cloned and the corresponding enzymes catalytic activity is under investigation.

These culture-independent approaches are designed to take advantage of the enormous diversity of soil microorganisms, aiming to isolate more efficient biocatalysts, either for the synthesis of chiral molecules, or for the biodegradation of pollutants.

Scientific news on this topic

Characterization of novel dioxygenases oxidizing PAHs in soil
Since most soil bacteria are uncultivable in the laboratory it is necessary to use molecular methods to learn on the potential of specialized species, like those that degrade PAHs in situ. By combining stable isotope probing (SIP) with an approach involving metagenomic sequencing of soil DNA, the genome of bacteria involved in the degradation of PAHs was partially revealed. From these data, we cloned four ring-hydroxylating dioxygenases, which exhibited original sequences and unusual catalytic activities towards PAHs.

Bacterial diversity and biodegradation of pollutants.
We have studied in this work soil bacteria that play a major role in the in situ biodegradation of polycyclic aromatic hydrocarbons. To identify such bacteria, we have implemented molecular methods, involving a culture-independent strategy.
[read more]

Biodegradation of a diesel additive.
We conducted a study of biodegradability of a diesel additive, 2-EHN, in which we isolated bacteria capable of degrading the synthetic compound whose effects on the environment and human health are unknown. We further elucidated the steps of the degradation of 2-EHN and identified the enzymes involved.
[read more - French version]

Bacterial enzymes attack the pollutants.
We have studied bacteria capable of breaking down toxic organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and we are interested in ring-hydroxylating dioxygenases that catalyze the oxidation of PAHs. This work can be used to design environmental biosensors.
[read more - French version]

Characterization of a dioxygenase which plays a key role in the degradation of PAHs.
We purified and characterized a dioxygenase that catalyzes the initial step of oxidation of polycyclic aromatic hydrocarbons (PAHs). Our work should lead to better understanding of what determines the specificity and catalytic properties of the enzyme. These studies may have biotechnological applications in the field of bioremediation of contaminated sites.
[read more - French version]

PhD theses

Florence Martin.
Bacterial diversity exploration in hydrocarbon polluted soil: Metabolic potential and degrader community evolution revealed by isotope labeling.
[On-line thesis]

Élodie Nicolau.
Biodegradation of 2-ethylhexyl nitrate by Mycobacterium austroafricanum IFP 2173.
[On-line thesis]


Harzallah B, Bousseboua H and Jouanneau Y
Diversity shift in bacterial phenol hydroxylases driven by alkyl-phenols in oil refinery wastewaters.
Environmental Science and Pollution Research, 2017, 24(16): 14376-14386

Jouanneau Y, Meyer C and Duraffourg N
Dihydroxylation of four- and five-ring aromatic hydrocarbons by the naphthalene dioxygenase from Sphingomonas CHY-1.
Applied Microbiology and Biotechnology, 2016, 100(3): 1253-1263

Chemerys A, Pelletier E, Cruaud C, Martin F, Violet F and Jouanneau Y
Characterization of novel polycyclic aromatic hydrocarbon dioxygenases from the bacterial metagenomic DNA of a contaminated soil.
Applied and Environmental Microbiology, 2014, 80(21): 6591-600

Kom Regonne R, Martin F, Mbawala A, Ngassoum MB and Jouanneau Y
Identification of soil bacteria able to degrade phenanthrene bound to a hydrophobic sorbent in situ.
Environmental Pollution, 2013, 180: 145-151

Martin F, Malagnoux L, Violet F, Jakoncic J and Jouanneau Y
Diversity and catalytic potential of PAH-specific ring-hydroxylating dioxygenases from a hydrocarbon-contaminated soil.
Applied Microbiology and Biotechnology, 2013, 97(11): 5125-5135

Esbelin J, Jouanneau Y and Duport C
Bacillus cereus Fnr binds a 4Fe-4S cluster and forms a ternary complex with ResD and PlcR.
BMC Microbiology, 2012, 12: art.125

Martin F, Torelli S, Le Paslier D, Barbance A, Martin-Laurent F, Bru D, Geremia R, Blake G and Jouanneau Y
Betaproteobacteria dominance and diversity shifts in the bacterial community of a PAH-contaminated soil exposed to phenanthrene.
Environmental Pollution, 2012, 162: 345-353

Jouanneau Y, Martin F, Krivobok S and Willison J
Ring-hydroxylating dioxygenases involved in PAH biodegradation: Structure, function and biodiversity.
In A-I Koukkou ed., Microbial bioremediation of non-metals: Current research, 2011, pp. 149-175, Caister Academic Press, Norfolk, UK

Schuler L, Jouanneau Y, Ni Chadhain SM, Meyer C, Pouli M, Zylstra GJ, Hols P and Agathos SN
Characterization of a ring-hydroxylating dioxygenase from phenanthrene-degrading Sphingomonas sp. strain LH128 able to oxidize benz[a]anthracene.
Applied Microbiology and Biotechnology, 2009, 83(3): 465-475

Schuler L, Ni Chadhain SM, Jouanneau Y, Meyer C, Zylstra GJ, Hols P and Agathos SN
Characterization of a novel angular dioxygenase from fluorene-degrading Sphingomonas sp. strain LB126.
Applied and Environmental Microbiology, 2008, 74(4): 1050-1057

Jakoncic J, Jouanneau Y, Meyer C and Stojanoff V
The catalytic pocket of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1.
Biochemical and Biophysical Research Communications, 2007a, 52(4): 861-866

Jakoncic J, Jouanneau Y, Meyer C and Stojanoff V
The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1.
Febs Journal, 2007b, 274(10): 2470-2481

Jouanneau Y, Micoud J and Meyer C
Purification and characterization of a three-component salicylate 1-hydroxylase from Sphingomonas CHY-1.
Applied and Environmental Microbiology, 2007, 73(23): 7515-7521

Jouanneau Y and Meyer C
Purification and characterization of an arene cis-dihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols.
Applied and Environmental Microbiology, 2006, 72(7): 4726-4734

Jouanneau Y, Meyer C, Jakoncic J, Stojanoff V and Gaillard J
Characterization of a naphthalene dioxygenase endowed with an exceptionally broad substrate specificity toward polycyclic aromatic hydrocarbons.
Biochemistry, 2006, 45(40): 12380-12391

Jouanneau Y, Willison JC, Meyer C, Krivobok S, Chevron N, Besombes JL and Blake G
Stimulation of pyrene mineralization in freshwater sediments by bacterial and plant bioaugmentation.
Environmental Science and Technology, 2005, 39: 5729-5735

Demaneche S, Meyer C, Micoud J, Louwagie M, Willison JC and Jouanneau Y
Identification and functional analysis of two aromatic ring-hydroxylating dioxygenases from a Sphingomonas strain degrading various polycyclic aromatic hydrocarbons.
Applied and Environmental Microbiology, 2004, 70: 6714-6725

Krivobok S, Kuony S, Meyer C, Louwagie M, Willison JC and Jouanneau Y
Identification of pyrene-induced proteins in Mycobacterium sp. 6PY1: Evidence for two ring-hydroxylating dioxygenases.
Journal of Bacteriology, 2003, 185: 3828-3841

Publications related to others topics

Nicolau E, Kuhn L, Marchal R and Jouanneau Y
Proteomic investigation of enzymes involved in 2-ethylhexyl nitrate biodegradation in Mycobacterium austroafricanum IFP 2173.
Research in Microbiology, 2009, 160(10): 838-847

Solano-Serena F, Nicolau E, Favreau G, Jouanneau Y and Marchal R
Biodegradability of 2-ethylhexyl nitrate (2-EHN), a cetane improver of diesel oil.
Biodegradation, 2009, 20(1): 85-94

Esbelin J, Jouanneau Y, Armengaud J and Duport C
ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus.
Journal of Bacteriology, 2008, 190(12): 4242-4251

Nicolau E, Kerhoas L, Lettere M, Jouanneau Y and Marchal R
Biodegradation of 2-ethylhexyl nitrate (2-EHN) by Mycobacterium austroafricanum IFP 2173.
Applied Environmental Microbiology, 2008, 74(20): 6187-6193