p-Hydroxylation reactions catalyzed by naphthalene dioxygenase

p-Hydroxylation reactions catalyzedbynaphthalenedioxygenaseKyoung Lee

Department of Microbiology, Changwon National University, Changwon, Kyongnam, Korea

Correspondence: Kyoung Lee, Department

of Microbiology, Changwon National

University, Changwon, Kyongnam 641-773,

Korea. Tel.: 182 55 279 7466; fax: 182 55

279 7460; e-mail:

[email protected]

Received 1 September 2005; revised 27

November 2005; accepted 28 November 2005.

First published online 21 December 2005.

doi:10.1111/j.1574-6968.2005.00079.x

Editor: Hans-Peter Kohler

Keywords

Rieske dioxygenase; hydroxylation; phenol;

hydroquinone.

Abstract

In this study, naphthalene dioxygenase is shown to catalyze the oxidation of

methylphenols and chlorophenols by p- and/or o-hydroxylation reactions. For

instance, m-cresol was oxidized to methylhydroquinone with formation of 3- and

4-methylcatechol as minor products. 2-Chlorophenol was exclusively oxidized to

chlorohydroquinone, which is an important building block for pharmaceutical

products and other organic compounds. The oxygen incorporated in the

p-hydroxylation reaction from m-cresol is derived from water with consumption

of O2.

Introduction

Naphthalene dioxygenase (NDO) from Pseudomonas

NCIB 9816-4 catalyzes the oxidation of naphthalene to

cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (naphtha-

lene cis-dihydrodiol) with the consumption of O2 and

NAD(P)H (Jeffrey et al., 1975). NDO is a multicomponent

enzyme system consisting of ReductaseNAP, FerredoxinNAP

and an a3b3 terminal oxygenase component, OxygenaseNAP

(Gibson & Parales, 2000). Electrons from NAD(P)H are

transferred through ReductaseNAP, FerredoxinNAP to the

oxygenase component. The oxygenase contains a Rieske

[2Fe–2S] redox center and mononuclear iron at the active

site, which is co-ordinated by a water, two histidines and one

bidentate aspartate forming a 2-His-1-carboxylate facial

triad (Que, 2000) involved in O2 activation and catalysis

(Kauppi et al., 1998; Karlsson et al., 2003). NDO has been

the subject of several structural and biochemical studies

(Kauppi et al., 1998; Wolfe et al., 2001; Karlsson et al., 2003;

Wolfe & Lipscomb, 2003), leading toward an understanding

of the mechanism for O2 activation and substrate hydro-

xylation in this superfamily of bacterial Rieske nonheme

iron oxygenases. These enzymes play an important role in

the environmental biodegradation of organic pollutants and

in the biotechnology industry for the production of value-

added chemicals (Hudlicky et al., 1999; Gibson & Parales,

2000; Boyd et al., 2001; Parales, 2003).

Naphthalene dioxygenase has a relaxed substrate specifi-

city and can oxidize almost 100 substrates. These include the

enantiospecific cis-dihydroxylation of polycyclic aromatic

hydrocarbons and the olefin groups of benzocycloalkenes,

benzylic hydroxylation, N- and O-dealkylation, sulfoxida-

tion and desaturation reactions (reviewed in Resnick et al.,

1996 and filed in The University of Minnesota Biocatalysis/

Biodegradation database http://umbbd.ahc.umn.edu). Re-

cently, it has been shown that NDO catalyzes oxidation of at

least 15 different indole derivatives to dyes, further advocat-

ing NDO as a versatile catalyst (Kim et al., 2003). In this

study, NDO is shown to catalyze mono-hydroxylation reac-

tions with phenols. These results show that phenols can be

important mechanism-probe substrates for NDO, and NDO

could be a useful biocatalyst for the hydroxylation of

phenolic compounds.

Materials andmethods

Biotransformationandanalyticalmethods

An Escherichia coli K-12 mutant CGSC7692 that does not

produce tryptophanase (tnaA5) was transformed with plas-

mid pDTG141 (Suen & Gibson, 1994), which expresses

NDO from Pseudomonas strain NCIB9816-4. This strain

was used in the biotransformation of phenol and its

derivatives. Escherichia coli CGSC7692(pDTG141) was cul-

tured in LB medium, and the NDO genes were induced with

isopropyl-b-D-thiogalactopyranoside as described pre-

viously (Suen & Gibson, 1994). The induced cells or E. coli

CGSC7692, used as a control, were suspended at an optical

FEMS Microbiol Lett 255 (2006) 316–320c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

density of one at 600 nm in 50 mM Na-phosphate buffer

(pH 7.0) containing 20 mM glucose. The reaction mixtures

(50 mL in 250 mL Erlenmeyer flasks) were incubated with

substrates (0.02%, volume in volume) for 48 h at 28 1C with

agitation at 180 r.p.m. At this point, the reaction mixtures

were centrifuged and the supernatants were extracted with

ethyl acetate, followed by concentration under reduced

pressure. Reaction products were identified by gas chroma-

tography/mass spectrometry (GC/MS) and thin layer chro-

matographic analysis as described previously (Kim et al.,

2003), and the results were compared with authentic com-

pounds. Phenols and dihydroxybenzenes were obtained

from Aldrich Chem. Co. (Milwaukee, WI).

Oxygen consumptionand18O2 incorporationexperiments

Oxygen consumption with purified NDO was measured at

24 1C with a Clark-type oxygen electrode (Rank Brothers,

Cambridge, England) as described previously (Lee & Gib-

son, 1996). Reactions were initiated by the addition of 1 mL

of a 100 mM solution of the substrate in methanol to a 1 mL

reaction vessel (final concentration 0.1 mM). The 18O2

incorporation experiment was carried out as described

previously (Lee & Gibson, 1996). The sealing of the reaction

bottles was confirmed by monitoring the 18O2 content in the

reaction headspace at initiation and termination of the

experiment. The 18O2 content in the reaction headspace is

indicated in the figure legend. The 18O contents in the

products were determined from GC/MS fragmentation

patterns.

Results anddiscussion

Escherichia coli CGSC7692(pDTG141) cells expressing NDO

catalyzed hydroxylation, with different regiospecificity, of

phenol, cresols and chlorophenols to yield dihydroxyben-

zenes as the end products. For instance, the dominant

products formed from phenol, o-, m-cresol, 2- and 3-

chlorophenol were hydroxyquinones by p-hydroxylation. In

contrast, p-cresol and 4-chlorophenol were predominantly

hydroxylated at the o-position, yielding 4-methyl- and

4-chlorocatechol, respectively. The results are shown in

Table 1.

Because the monohydroxylation of phenols by NDO has

not been observed previously, the reaction rate and the

requirement of O2 in the reactions were further investigated

using purified NDO. In oxygen consumption experiments,

the reaction rates of oxygen consumption were not notably

different for o-, m- and p-cresol, but the reaction was

significantly reduced in the presence of phenol (Fig. 1). It is

also noted that the extent of uncoupling detected from the

baseline was not significant in the absence of the substrate

added. After 30 min, the analysis of the reaction mixtures

yielded hydroquinone (45%), methylhydroquinone (98%),

methylhydroquinone (81%) and 4-methylcatechol (1%) as

the dominant products from phenol, o-, m- and p-cresol,

respectively. In all these reactions, more O2 was consumed

than substrate added, indicating that the O2 consumption

was partially uncoupled from product formation. The origin

of incoming oxygen in the oxidation of phenol and m-cresol

was further investigated with purified NDO in the presence

of 18O2 (Fig. 2). Interestingly, the oxygen incorporated in

Table 1. Products formed from phenols by whole cell transformation with Escherichia coli CGSC7692(pDTG141) expressing naphthalene

dioxygenase�

Substrate

GC/MS

Product(s) Mass Retention time (min) Relative yield (%) TLCw Rf

Phenol Catechol 110 10.65 21.5 0.32

Hydroquinone 110 11.66 57.6 0.18

o-Cresol 3-Methylcatechol 124 11.48 5.5 0.41

Methylhydroquinone 124 12.64 46.8 0.25

m-Cresol 3-Methylcatechol 124 11.48 2.9 0.41

4-Methylcatechol 124 12.03 11.6 0.34

Methylhydroquinone 124 12.64 30.8 0.25

p-Cresol 4-Methylcatechol 124 12.03 32.7 0.34

2-Chlorophenol Chlorohydroquinone 144 12.57 88.0 0.30

3-Chlorophenol Chlorohydroquinone 144 12.57 16.2 0.30

4-Chlorocatechol 144 14.20 3.5 0.27

4-Chlorophenol 4-Chlorocatechol 144 14.20 26.2 0.27

�Experimental conditions are described in Materials and methods. Ninety-seven percent conversion of naphthalene into naphthalene cis-dihydrodiol

was observed under the same experimental conditions. The control experiments with E. coli CGSC7692 did not yield detectable levels of products.wChloroform–acetone (9 : 1) was used as a developing solvent.

GC/MS, gas chromatography-mass spectrometry; TLC, thin layer chromatography.

FEMS Microbiol Lett 255 (2006) 316–320 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

317Phenol oxidation by naphthalene dioxygenase

hydroquinone and methylhydroquinone, formed from phe-

nol and m-cresol, respectively, contained 10.2% and 0% of

the normal enrichment of 18O, respectively. This result

indicated that the oxygen in the p-hydroxylated products

predominantly came from H2O. Under the same experi-

mental conditions, NDO completely incorporated 18O2 into

naphthalene to form naphthalene cis-dihydrodiol (Fig. 2).

Monohydroxylation of phenols has been reported pre-

viously for bacterial Rieske nonheme iron dioxygenases

such as toluene dioxygenase (Spain et al., 1989), biphenyl

dioxygenase (Seeger et al., 2001) and 2,4-dinitrotoluene

dioxygenase (Keenan et al., 2004). For instance, toluene

dioxygenase catalyzes the oxidation of phenol and 2,5-

dichlorophenol to catechol and 3,6-dichlorocatechol, re-

spectively. It was suggested that the latter product was

formed by dioxygenation of the aromatic nucleus followed

by elimination of water. Although 2,4-dinitrotoluene dioxy-

genase and its V350 variants hydroxylated phenols at the

para position, the detailed reaction mechanism has not been

examined (Keenan et al., 2004).

The findings that incoming oxygen in the para-hydro-

xylation reaction is derived from water could be accounted

for by two-electron oxidation of the phenol nucleus and

concomitant recombination of hydroxide from water. Based

upon previously proposed cis-dihydroxylation pathways

(Wolfe et al., 2001; Karlsson et al., 2003; Wolfe & Lipscomb,

2003), the catalytic mechanism for the oxidation of m-cresol

by NDO can be depicted as in Fig. 3. For instance, m-cresol

can bind at the active site with the hydroxyl group toward

iron, as observed previously in the crystal structure of

naphthalene cis-dihydrodiol binding to NDO (Karlsson

et al., 2003). In this case, the mononuclear iron serves as a

Lewis acid and oxygen in m-cresol serves as a Lewis base,

forming an iron-phenoxide intermediate. In NDO catalysis,

the involvement of a (hydro)peroxo intermediate has been

proved, as hydrogen peroxide is released during the ben-

zene-based uncoupling reaction (Lee, 1999) and hydrogen

peroxide is an oxidant for a single turnover experiment

(Wolfe et al., 2001). Thus, the transient ternary complex

with mononuclear iron-peroxide-m-cresol could be pre-

dicted. For NDO catalysis, the concerted action of FeIII-

OOH has been proposed as being energetically favorable for

the cis-dihydroxylation reaction (Karlsson et al., 2003;

Bassan et al., 2004), and evidence for the heterolytic cleavage

of FeIII-OOH leading to the formation of an HO-FeV = O

intermediate has also been presented for the biomimetic

systems (Chen & Que, 2001). At present, it is unknown

which intermediate can abstract two electrons from the

nucleus of m-cresol, although the formation of an FeV = O

intermediate is plausible as proposed in Fig. 3. In a previous

Substrate

NADH

[O2]

(10

0µM

)

A

CD

E

B

3 min

Fig. 1. Oxygen consumption by naphthalene dioxygenase in the pre-

sence of naphthalene (A), phenol (B), o-cresol (C), m-cresol (D) and p-

cresol (E). The additions of NADH and substrate are indicated by arrows.

(a)

(b)

(c)

120

90

60

30

0

120

90

60

30

0

20

10

0

109

122

162 163 164 165 166 167

123 124 125 126

110 111 112

m /z

Rel

ativ

e in

tens

ity (

%)

Fig. 2. Mass spectra of hydroquinone (a), methylhydroquinone (b) and

naphthalene cis-dihydrodiol (c) in the molecular ion region. The sub-

strates were phenol, m-cresol and naphthalene, respectively. Open bars

are from natural atmospheric conditions and filled bars from 90%, 90%

to 87% 18O2 from total O2, respectively.


318 K. Lee

study, it was shown that NDO can catalyze alcohol oxidation

and desaturation reactions, which involve an overall two-

electron oxidation without the incorporation of oxygen (Lee

& Gibson, 1996; Resnick et al., 1996). Currently, it is not

clear whether all these reactions result from the same or

different active oxygen species. Furthermore, the partial

incorporation of 18O2 into phenol may be due to the

difference in the position of the nucleus of phenol at the

active site, e.g., hydroxyl group being away from mono-

nuclear iron, leading to typical hydroxylation, which incor-

porates 18O2 into the substrate (Lee & Gibson, 1996). But

the detailed mechanism awaits further investigation with

biophysical and crystallographic studies.

In summary, NDO has been shown to catalyze the mono-

hydroxylation of phenols to yield dihydroxybenzenes. Oxy-

gen in the products of p-hydroxylation was derived from

water with oxygen consumption. The reactions can be

exploited to probe the O2 activation mechanism of NDO

because of the involvement of uncoupling in catalysis with

readily soluble substrates. In addition, these reactions can be

developed to produce commercially important chemicals

such as hydroquinones via an environmentally benign

biotransformation process.

Acknowledgements

This research was financially supported by Korea Research

Foundation Grant (KRF 2003-005-I00061). The author

would like to thank Prof. David T. Gibson (University of

Iowa) for motivating this research and for discussions, and

Dr Koo Lee (LG Life Sciences) for proposing the reaction

mechanism.

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O HFeIII

OO H

OO

His-NHis-N

Asp

FeII

O

FeV O

OH− e−+H+

O2+e−+H+

m-Cresol

OH−

O

FeIII O

HO

OH

OHH

H+

+

Methyl-hydroquinone

H3C

H2O

H3CH3C

H3C

Fig. 3. Proposed catalytic mechanism for

m-cresol oxidation by naphthalene dioxygenase.

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320 K. Lee

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p-Hydroxylation reactions catalyzed by naphthalene dioxygenase