Bacterial Pathways for Degradation of tics

8/9/2019 Bacterial Pathways for Degradation of tics

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HIGHLIGHT www.rsc.org/npr | Natural Product Reports

Bacterial pathways for degradation of nitroaromatics

Zoe C. Symons and Neil C. Bruce*

Received (in Cambridge, UK) 27th June 2005

First published as an Advance Article on the web 6th October 2006

DOI: 10.1039/b502796a

Covering: up to 2006

The last one hundred years have seen a massive expansion in the chemicals industry; however, with this

progress came the concomitant pollution of the environment with a significant range of xenobiotics.

Nitroaromatic compounds form one such category of novel environmental contaminants and are

produced through a large number of industrial processes, most notably the pesticides, dyes and

explosives industries. Whilst singly nitrated aromatic compounds are usually mineralised in the

environment, multiply nitrated aromatics, such as the explosive 2,4,6-trinitrotoluene (TNT), are

recalcitrant and highly toxic. The predominant route of biological transformation of aromatic

compounds is oxidation; however, the presence of three electron-withdrawing nitro-groups around the

ring prevents oxidation, rendering such compounds resistant to biodegradation. The subsequent

accumulation of these contaminants has stimulated much research leading to the isolation of bacteriathat possess, to varying extents, the ability to remediate explosives and other nitroaromatic pollutants.

The extreme environments created by these toxic substances accelerate the evolutionary process and

examples of bacteria that have conscripted metabolic enzymes for novel remediatory pathways are

included. This Highlight ends with a discussion of the future of nitroaromatic bioremediation including

engineering plants to express bacterial enzymes for use in bioremediation programs.

Introduction

The chemical versatility of the nitrogroup means nitroaromatic

compounds are widely used in industry in the synthesis of

dyestuffs, pesticides, explosives and polyurethane foams. Ni-

troaromatics also form the basis of explosives such as 2,4,6-

trinitrotoluene (TNT) and 2,4,6-trinitrophenol (picric acid)and have historically been produced on a massive scale. 2,4-

CNAP, The Department of Biology, AREA 8, The University of York, POBox 373, York, England YO10 5YW. E-mail: [email protected]; Fax: 44-(0)1904 328801; Tel: 44-(0)1904 328777

Zoe Symons obtained her Master of Biochemistry from the University of Bath, studied for her PhD at the Institute of Biotechnology,

University of Cambridge, and is currently a post-doctoral research fellow in CNAP, University of York.

Zoe C. Symons Neil C. Bruce

Neil Bruce is currently Professor of Biotechnology at CNAP in the

Department of Biology at the University of York. Following a PhDin Microbial Biochemistry at the University of Kent in 1987, he

joined the Institute of Biotechnology at the University of Cambridge

as a postdoctoral research associate with Prof. Chris Lowe and was

subsequently awarded a Research Fellowship at Wolfson College. He

was appointed to a University Lectureship in Biotechnology in the same

departmentin 1990 and subsequently appointed to a Fellowship at Trinity

Hall. He was promoted to a Personal Readership in Biotechnology in

2001. In 2002 he was appointed to the Chair of Biotechnology at the

University of York. The major research themes of his laboratory are

microbial metabolism, biocatalysis and environmental biotechnology.

Dinitrotoluene (DNT), an intermediate in TNT manufacture

and starting material for the synthesis of toluene diisocyanate,

is also produced on a large scale. The extensive use of many

nitroaromatic compounds in such chemical processes has led to

their widespread contamination of the environment. Five hundred

thousand U. S.gallons of water, contaminated with trinitrotoluene

and other nitroaromatics, may be released into the environment

by one TNT-manufacturing plant in one day.1 At some munitions

manufacturing and processing sites the contamination can be

as high as 200g TNT per 1 kg of soil.2 Many of the multiply-

nitrated compounds are cytotoxic and have been classified as

probable carcinogens. TNT, for example, is documented as causing

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erythrocyte abnormalities, dysfunction of the liver and cancer

in mammals. Unfortunately, once released into the environment

many of these compounds persist for long periods of time.

This is likely to be because similar compounds rarely occur

in the Natural world, their structural novelty rendering them

resistant to breakdown as indigenous organisms have no means of

metabolising them.3

The inherent toxicity of these anthropogenic compounds means

they should not be permitted to remain in the environment;

however, the widespread contamination of the soil and ground

water worldwide means that inexpensive and sustainable tech-

nologies to remove these toxic compounds need to be developed

and employed. Current methods of remediating contaminated

sites, such as incineration and composting, can be expensive

and harmful. Incineration of soil to rid it of explosives can

result in the exposure of workers to high levels of toxins.3,4,5 One

solution to the problem of nitroaromatic contamination, which

is considered both economically feasible and environmentally

sound, is bioremediation. This is the use of organisms, such as

microbes or plants, to degrade or detoxify hazardous materials. To

these ends research groups have employed selective enrichments,

with the nitroaromatic supplied as the sole carbon and/or nitrogen

source for growth, to isolate specialist microbes with the ability

to transform nitroaromatics. In this way, bacteria and fungi have

been isolated with the ability to transform nitroaromatic com-

pounds. Enzymes responsible for conferring the nitroaromatic-

transforming activity on a number of microbes have been cloned

and characterised, offering an enhanced understanding of the bac-

terial metabolism of nitroaromaticsthe subject of this Highlight.

Enzymes associated with nitroaromatic transformation

Many enzymes have been characterised as being capable of trans-

forming nitroaromatic compounds via oxidative and reductiveattack. These include monooxygenases, dioxygenases, hydride

transferases and nitroreductases. Most recently, an iron-only

hydrogenasefrom Clostridium acetobutylicum hasalso been shown

capable of reducing TNT to its dihydroxylamino derivative in a

hydrogen-dependent manner and has been demonstrated to be

responsible for the great extent of the nitroreductase activity ofC.

acetobutylicum.6

The evolution of nitroaromatic degradation pathways

Recent work on the biodegradation of dinitrotoluene by

Burkholderia cepacia R34 offers one of the most elegant examples

of how bacteria have evolved to not only tolerate but to metabolise

nitroaromatic compounds as a nutrient source for growth.7 B.

cepacia R34 was isolated from surface water from an ammunition

plant and was therefore subject to significant environmental selec-

tive pressures. Analysisof theenzymes it employs to transform2,4-

DNT has revealed that the bacterium has co-opted enzymes from

diverse pre-existing pathways to produce one specific to 2,4-DNT.7

The genes encoding the enzymes required for degradation are

found on a contiguous 27 kb region of DNA, which also includes

regions devoted to the regulation of the pathway. Considering that

2,4-DNT has only been produced for the last one hundred years

the ability of bacteria to piece together a new metabolic pathway

to utilise this compound as a carbon source for growth is quite

remarkable.

The enzymes which form the pathway have been characterised

individually and their reactions are illustrated in Fig. 1.

Fig. 1 The metabolism of 2,4-dinitrotoluene catalysed by Burkholderia

cepacia R34, adapted from Johnson et al.7 Compounds in brackets remain

hypothetical.

The first denitration is catalysed by DNT dioxygenase (encoded

by dntAaAbAcAd), a Rieske non-haem iron oxygenase recruited

from a naphthalene degradation pathway. This oxygenase has

three parts, an oxidoreductase (Aa), an iron-sulfur ferredoxin pro-

tein (Ab) and a terminal oxygenase centre (Ac and Ad form a and

b subunits). The second denitration is catalysed by the 4-methyl-

5-nitrocatechol monooxygenase (dntB) believed to be seconded

from a degradation pathway originally for chloroaromatic com-

pounds. The 2,4,5-trihydroxytoluene then undergoes ring fission

catalysed by 2,4,5-trihydroxytoluene oxygenase (dntD). The 2,4-

dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid, the ring fission

product, is metabolised by the enzyme dntG to yield pyruvate and

methylmalonic acid semialdehyde in turn transformed by dntE,

although the final product propionyl-CoA remains hypothetical.

All the last three enzymes have been hijacked from amino acid

846 | Nat. Prod. Rep., 2006, 23, 845850 This journal is The Royal Society of Chemistry 2006


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degradation pathways. The enzymes which form this novel 2,4-

DNT degradation pathway are all encoded on a single contiguous

length of DNA, producing a pathway specific to an anthropogenic

compound.

The correct functioning of a pathway that has adapted or been

created to transform nitroaromatic compounds relies not only

on possessing enzymes with the appropriate catalytic activity but

also on the expression of these enzymes in the presence of the

nitroaromatic compound. The first example of a nitroaromatic-

triggered lysR transcriptional activator was reported recently.8

Two nitroarene dioxygenases were studied, nitrobenzene dioxyge-

nase (NBDO) in Comomonas sp. strain JS765 and 2-nitrotoluene

dioxygenase (2NTDO) from Acidovorax sp. strain JS42. They are

under the control of identical LysR related proteins, NbzR and

NtdR, homologous to the previously reported NahR.

The work revealed the expression of 2NTDO and NBDO can

be induced by a plethora of nitroaromatic compounds including

nitrobenzene, 2-, 3- and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene

and aminodinitrotoluenes in addition to the endogenous salicylate

and anthranilate. The metabolism of these compounds was not

necessary for induction suggesting the nitroaromatic compounds

themselves are the actual effector molecules with the NtdR

essential for induction. This represents a pathway in evolution.

Enzymes capable of transforming nitroaromatic compounds are

being organised to be expressed in response to the presence

of these novel substrates, although at present the regulatory

proteins still respond to their endogenous effectors salicylate and

anthranilate.

Nitrogen assimilationby bacteria has also been studied with ref-

erence to TNT degradation. Recent work employing Pseudomonas

putida JLR11 has shown that the nitrogen containing products

released on TNT degradation are assimilated via the glutamine

synthetaseglutamate synthase (GSGOGAT) pathway.9

The metabolism of naturally occurring nitroaromatic

compounds

Very few naturally occurring nitroaromatic compounds are

known. Recently, for the first time, the degradation pathway

of one of these compounds, 3-nitrotyrosine, a product of the

interaction between reactive nitrogen species and amino acids,

was elucidated.10 Using 3-nitrotyrosine as sole growth substrate in

soil enrichment cultures, multiple bacteria were isolated capable

of catalysing the degradation of 3-nitrotyrosine with the release

of nitrite and ammonia. Two species, Burkholderia sp. strain

JS165 and Variovorax paradoxus JS171 were chosen for further

study.

The pathway for the breakdown of 3-nitrotyrosine was found

to be both separate from the tyrosine degradation pathway and

inducible in its entirety by cell growth on 3-nitrotyrosine or

its breakdown product 4-hydroxy-3-nitro-phenylacetate (HNPA)

(Fig. 2).

The pathway starts with an a-ketoglutarate dependent deam-

ination to produce the conjectural intermediate 4-hydroxy-

3-nitrophenyl pyruvate. This is decarboxylated to yield HNPA,

in turn denitrated to homoprotocatechuate by a monooxygenase.

The homoprotocatechuate is cleaved by homoprotocatechuate-

2,3-dioxygenase to yield 5-carboxymethyl-2-hydroxy-cis,cis-

Fig. 2 The proposed pathway for the breakdown of the naturally occur-

ring nitroaromatic compound 3-nitrotyrosine, adapted from Nishino and

Spain.10 Compounds in brackets are proposed intermediate compounds.

muconic semialdehyde, the metabolism of which has been

thoroughly characterised. This pathway utilised for 3-nitrotyrosine

metabolism has been demonstrated to be analogous to the tyro-

sine degradation pathway, which also proceeds via homoproto-

catechuate, and may have derived from the tyrosine pathway.

Nitroreductases

The enzymatic transformation of triply nitrated aromatic com-

pounds, such as the explosives TNT and picric acid, is gener-

ally limited to reductive transformations. Enzymes capable of

catalysing such reactions are exemplified by the Enterobacter

cloacae PB2 enzyme, PETN reductase (PETNr), a monomeric

40 kDa flavoenzyme,11,12 which is capable of catalysing both the

nitroreduction of TNT and the ring reduction, as illustrated in

Fig. 3.

PETNr was isolated through its ability to confer resistance

to PETN, a nitrate ester explosive, on the bacterium.11 PETNr

was found to be a member of the Old Yellow Enzyme family

of flavoenzymes and was the first of a series of enzymes from



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Fig. 3 The biotransformation of 2,4,6-trinitrotoluene as catalysed by

PETN reductase from Enterobacter cloacae PB2.

this family that have since proven capable of transforming

explosives.1216 PETNr is capable of reductively liberating nitrite

not only from PETN and nitroglycerine, but from nitroaromatic

explosives such as TNT and picric acid. The nitroreduction of

triply nitrated aromatics (Fig. 3, Pathway A) is more common.

Many bacteria/enzymes have proven capable of opportunistic

nitroreduction of nitroaromatic compounds. A recent example

is that of PnrA,17 an NADPH dependent nitroreductase, ho-

mologous to nfsA, the Escherichia coli nitroreductase, capable

of transforming TNT to yield 4-hydroxylamino-2,6-DNT via a

ping-pong bibi mechanism. It is capable of reducing multiple

nitroaromatic substrates including 2,4-DNT, 3-nitrotoluene, 3-

and 4-nitrobenzoate, 3,5-dinitrobenzamide and 3,5-dinitroaniline,

although it was most efficient with TNT.

The nitroreduction of TNT results in the production of

metabolites that are still toxic. Hydroxylamino-dinitrotoluenes

(HADNTs) damage DNA causing mutations18 and result in

the redox cycling of the TNT derivatives with the concomitant

production of harmful reactive oxygen species.19 This has been

put to good use in the field of cancer therapy as discussed later.

Interestingly, the nitroso intermediate inherent in this pathway

has not been isolated. Recent work testing the nfsB encoded

E. coli nitroreductase against nitroso- and nitro-toluene and

benzene (Fig. 4) demonstrated that the nitrosoderivatives are

far better substrates for the enzyme, having specificity constants

2030 000 times that of the nitro-substrates.20 This offers an

explanation as to why the nitrosoderivatives of these and other

nitrocompounds are seldom seen in biotransformations.

Fig. 4 The structures of nitro- and nitroso-toluene and benzene.

Hydride transferases

A second reductive biotransformationof TNT (Fig. 3, Pathway B)

has been observed,which offers hope for the complete degradation

of TNT. It is catalysed bysome members of theOld YellowEnzyme

family16,21,22 including PETNr. This second pathway features the

reduction of the aromatic ring of TNT. Aromatic transformations

are intuitively associated with oxidative attack due to the high

electron density of the benzene ring; however, the three NO2groups present in TNT are sufficiently electron-withdrawing to

render the ring electron-poor and subject to reductive attack. The

result is the production of hydride- and dihydride-Meisenheimer

TNT complexes (H-TNT and 2H-TNT) and hence the loss

of aromaticity. The ultimate products of the pathway are not

known; however, the reduction of the dihydride-Meisenheimer

TNT complexes has been confirmed to be enzyme-catalysed,23 and

in the course of the transformation, nitrite is released. The final

transformation products remain elusive, although recent work has

characterised them as lacking visible absorbance and being highly

polar. They contain less nitrogen than TNT, are non-aromatic and

water soluble, all of which suggests that they willbe less recalcitrant

to degradation and less toxic than both TNT and the products of

the nitroreduction pathway.5,16

A second example of such a hydride transfer reaction with a

nitroaromatic compound comes in the form of picric acid degra-

dation by Rhodococcus (opacus) erythropolis and Nocardiodes

simplex FJ21A, reviewed in Heiss and Knackmuss24 (Fig. 5).

Picric acid is more easily degraded than TNT. Substantial

work characterising the entire pathway of picric acid degradation

has been performed by the Knackmuss group in their work on

Rhodococcus (opacus) erythropolis HL PM-1 and Nocardioides

simplex FJ21A.2427 The initial reaction is hydride addition to

the aromatic ring, to create a hydride Meisenheimer complex,

as discussed with TNT; however, with picric acid no nitrore-

duction is observed, only the hydride addition as catalysed

b y a F420

-dependent hydride transferase (NpdI) and its F420

reductase (NpdG).25,27 A second hydride addition is catalysed by

NpdC/NpdG in R. (opacus) erythropolis; whereas, in N. simplex,

both hydride transfers are catalysed by the same enzyme.

Following the production of the dihydride Meisenheimer com-

plex it is hypothesised that the multiple products observed are due

to the tautomerisation of the protonated dihydride Meisenheimer

complex, catalysed by NpdH.27 The hydride Meisenheimer com-

plex of 2,4-dinitrophenol has been identified as an intermediate

of picric acid degradation in Nocardioides sp. strain CB 222,

which would suggest that nitrite is released from the dihydride

Meisenheimer complex or one of its protonated tautomers.24 Inter-

estingly, R. erythropolis can also catalyse the ring hydrogenation



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Fig. 5 The transformation of picric acid by Rhodococcus (opacus)

erythropolis HLPM1, adapted from Heiss and Knackmuss.24

of TNT, similar to the reaction catalysed by PETNr; however,

unlike PETNr, in spite of the production of 2H-TNT, no nitrite

release is observed.28

Bacterial enzymes applied

It seems that the most widespread source of nitroaromatic pollu-

tion is from the explosives industry, either through the active use

of explosives or through the manufacture and decommissioning of

munitions. Having characterised bacteria capable of transforming

and degrading explosives it seems a logical next step to use these

bacteria to remove the toxic explosives from soil. There are,

however, disadvantages in using bacteria for this purpose. Firstly,

when in competition with the local bacterial ecosystem, in non-

optimal growth conditions, there may be a significant drop in

the biomass of the applied bacteria. Equally, co-metabolism can

necessitate the addition of extra substrates to induce the enzyme

required for pollutant transformation.29 Additionally, although

many strains of explosives-detoxifying microorganisms have been

isolated from contaminated soil, pollution remains a problem

at these sites. This can be overcome using genetic engineering;

however, overwhelming anti-GMO public opinion makes such

a course of action difficult with genetically modified microbes.

Considerable research has been devoted to the phytoremediation

of explosives, that is usingplants to biotransform explosives. Plants

are uniquely suited to the removal of explosives contamination

due to their penetrating root systems, comparably high biomass

and compatibility with restorative ecology. Original work sought

to employ genetically unmodified plant species in this process;

however, major disadvantages were the inherently low levels of

detoxification coupled with the susceptibility of these plants to

the toxicity of the explosives. For phytoremediation to be a com-

petitive alternative to current methods of explosives degradation,

plants need to efficiently take up the contaminants and break

them down or transform them to less toxic intermediates. This has

been achieved by the constitutive expression of bacterial enzymes

in plants.30 The expression of both nitroreductase and PETN

reductase from Enterobacter cloacae PB2 has been achieved in

arabidopsis and tobacco plants.The resultant PETNr plants could

survive in concentrations of GTN and TNT (1 mM and 0.05 mM

respectively) that were toxic to genetically unmodified tobacco

plants.30 Similarly, aromatic nitroreductases, when expressed in

plants, provided even greater resistance to the toxic effects of

TNT.31

Bacterial enzymes are similarly being used outside their natural

environment in novel cancer therapies. Most particularly E. coli

oxygeninsensitive nitroreductase (nfsB)is beingused to activate ni-

troaromatic compounds, such as the dinitrobenzamide prodrugs,

to their cytotoxic hydroxylamino derivatives to destroy tumours.

Mammalian nitroreductases do not catalyse the activation of these

compounds, so the bacterial nitroreductases, when targeted to

the tumour using ADEPT (antibody directed enzyme prodrug

therapy) or GDEPT (gene-directed enzyme prodrug therapy),

exclusively activate the compounds in the vicinity of cancerous

growth. Recent determination of the nitroreductase structure in

complex with a series of these dinitrobenzamide drugs now paves

the way not only for the rational design of novel prodrugs but

also for the rational design of the enzyme specifically for cancer

therapies.32

Conclusions

An understanding of the bacterial transformation of nitroaro-

matic compounds has clear benefits for bioremediation research,

medicine and industry. In addition to a more detailed understand-

ing of how the initial nitroaromatic oxidases or reductases operate,

an enhanced appreciation of the role of enzymes that function

downstream of these initial attack enzymes will facilitate the

optimisation of remediation pathways through the upregulation ofkey downstream metabolic enzymes. In addition to a contribution

to the field of bioremediation, the elucidation of dissimilation

pathways for nitroaromatic compounds is likely to continue to

furnish a rich source of enzymes as biocatalysts for the synthesis

of high value chemicals and drugs.

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Bacterial Pathways for Degradation of tics

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