hydroxylation of 4-chlorobenzoate by an Arthrobacter sp

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 2, 1984 October 30, 1984

Pages

669-674

THEORIGIN OF THEOXYGEN INCORPORATED DURINGTHE DEHALOGENATION/ HYDROXYLATION OF 4-CHLOROBENZOATE BY AN ARTHROBACTER SP T.S. Marks',

R. Wait*, A.R.W. Smith8 and A.V. Quirk'

'Microbial Technology Laboratory PHLS Centre for Applied Microbiology h Research Porton Down, Nr Salisbury Wilts SP4 OJG U.K. 2Bacterial Metabolism Research Laboratory PHLS Centre for Applied Microbiology h Research Porton Down, Nr Salisbury Wilts SP4 OJG U.K. 3School of Biological Sciences and Environmental Health ThamesPolytechnic London SEi8 6PF U.K. Received September 27, 1984 An Arthrobacter sp. has been shown to deha yielding 4-hydroxybenzoate. Experiments with presence of cell-free extracts, the hydroxyl group which is substituted onto the aromatic nucleus during dehalogenation is derived from water and not from molecular oxygen. Dehalogenation therefore is not catalysed by a mixedfunction oxidase; instead a novel aromatic hydroxylase is implicated in the 0 1984 Academic Press, Inc. reaction. The dehalogenation of haloaliphatic widely

studied (1,&j)

compoundsby aerobic bacteria has been

and the displacement of the halogen by a hydroxyl

has been characterised

as a hydrolytic

process (4,5).

The corresponding

dehalogenation of the aromatic nucleus has been occasionally The

reaction

mechanisms

dehalogenation/hydroxylation aromatic oxygenases. acid by an aromatic Similarly,

studied

thus

far

that

(6,7).

aromatic

metabolism by known

For example, the dehalogenation of 4-chlorophenylacetic dioxygenase

in a Pseudomonas sp. has been reported

the dehalogenation of 4-fluorophenylalanine

phenylalanine

reported

suggest

may be due to fortuitous

group

4-monooxygenase (EC 1.14.16.1)

(8).

to produce tyrosine

purified

from rat

(NCIB lZOl3),

isolated

by

and sheep liver

has been described 19X An Arthrobacter sludge inoculum, chlorobenzoate

sp., strain

TM-l

from a sewage

has been shown to be capable of the dehalogenation

as the first

step in the degradation of this

of 4-

compound (10,111. 0006-291X/84 $1.50

669

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 124, No. 2, 1984 The

product,

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS is

A-‘hydroxybenzoate,

subsequently

metabolized

via

protocatechuate.

I-Chlorobeneoate

Preliminary free at

extracts low

oxygen

catalysed were

studies

substituted

4-chlorobenzoate

function

performed the

sp.

indicated

oxygenase to

aromatic

ring,

that result

(11).

determine

Protocatechuats

dehalogenation

an unexpected

concentrations;

onto

4-chlorobenzoic

on the

of the Arthrobacter

by a mixed

therefore

I-Hydroxybenzoate

the

maximum if

the

Labelling source during

system activity

the

occurred

dehalogenation

experiments of

in cell-

the

was

using

hydroxyl

‘00 group

dehalogenation

of

acid. I(IKl’DODS

MATEl?IALSAND

The growth of the Arthrobacter sp. and the preparation of cell-free The extracts extracts by freeze-pressing, were performed as Marks et al. (11). were inc bated with 4-chlorobenzoate in the presence of a source of labelled All experiments were performed in duplicate. oxygen C“01 as follows. Labelled

Water

The cell-free supernatant was incubated in 50 mM potassium phosphate buffer pH 7.0, containing 5 mM dithi threitol (DTT) and 1 mM A-chlorobenzoate. This buffer contained 40% (v/v) H 1%0 (95% enrichment, Miles Biochemicals, Stoke Poges, UK). Buffer (2 ml) an ?I cell-free extract (0.5 ml), in a Thunberg tube, were degassed by evacuation and flushed with nitrogen three times prior to mixing and incubation. To determine whether hydroxyl exchange between 4-hydroxybenzoate and H2’*0 had occurred, experiments were performed in which the 4-chlorobenzoate was replaced by 1 mM 4-hydroxybenzoate in the incubation mixture. Labelled

Oxygen

The cell-free supernatant was incubated in 50 q M potassium phosphate buffer pH 7.0, containing 5 mM DTT and 1 mM 4-chlorobenzoate. Buffer (2 ml) and cell-free supernatant (0.5 ml), in a Thunberg tube, were twice degassed by evacuation and flushed with nitrogen. The tub78 were then evacuated again and filled with oxygen gas containing 50% (v/v) O2 (Miles Biochemicals, Stoke Poges, UK), prior to mixing and incubation. Incubation In all the experiments the Thunberg tubes were incubated at 25OC for 200 minutes. The reaction was terminated by the addition of 0.5 M HCl (10 ml). The acidified mixture was twice extracted with diethyl ether (AR. Grade, May & 670

Vol. 124, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Baker Ltd., Dagenham, UK) and the combined ether phases were evaporated to dryness in a stream of air. The extracts were then methylated using diazomethane, as described by Vogel (12). The methylated extracts were analysed as below. Gas liquid

chromatography/Uass

spectrometry

Gas chromatography/mass spectrometry was performed with a DuPont 21-491 double focussing mass spectrometer (DuPont Instruments, Stevenage, UK), interfaced to a Varian 2700 series chromatograph (Varian Associates, Walton-onThames, UK) via a single stage glass jet separator. The chromatograph was equipped with a 2 m x 2 mm silanised glass column, packed with 3% SE30 on 100/120 mesh s pelcoport (Supelco., Bellefonte, USA). Helium, at a flow rate of 30 cm3 min’ Y was used as carrier gas. The injector and detector ovens were maintained at 250‘0 C, rhilst the column oven was temperature programmed from 120 - 200°C at 10°C min’ . Ionisation was effected by electron impact at an ionisation energy of 70eV. The instrument was calibrated over the range 27 - 617 atomic mass units scanned at 2 seconds per decade. A DuPont and the magnet was repetitively 21-0948 dual disc data system (DuPont Instruments, Stevenage, UK) was used for mass assignment and data reduction.

REEXJLTS Labelled

Water

When the H2’S0,

extract

approximately

contained peaks

cell-free

labelled

2 mass units

hydroxybenzoate

without

ion,

cell-free

incorporation

1.5

pmole

oxygen

(Fig.

1).

than

those

heavier (Fig.

hydroxybenzoyl

was incubated

2)

of 4-hydroxybenzoate This

is

produced

mlz

at

H0.C6H4.&). extract,

with

152

was formed,

demonstrated

the

(molecular

presence

by the

by authentic

When 4-hydroxybenzoate in

4-chlorobenzoate

of

and 40% of

which

detection

methylated ion)

and

H2180,

of

[160]-4-

m/z

was incubated 40% (v/v)

37%

121

(4-

with

or

no

label

was observed.

COOMe + ;;’ Dl I[o1 I %L?.L&A OH

0

OH

Figure

1

Mass ‘spectrum dehalogenation

of methylated of kchlorobenzoate

671

4-hydroxybenzoate in the

presence

producej8by of Hz

enzymic 0.

BIOCHEMICAL

Vol. 124, No. 2, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100 80

Figure

Labelled

Mass spectrum

2

the

approximately for

the

121

were

cell-free 0.6

extract

umole

not

of label

accompanied

presence

was

one labelled

which

ions, gave

of two

would

have

only

170 and 172 and m/z

168 and

single

peaks

oxygen

3

Mass spectrum dehalogenation

and there

heavier.

further

(v/v)

m/z

the

ion)

at m/z

atoms by triple

peaks

being

137 and 139

Authentic

methylated

137.

protocatechuate

was by the

and m/z

168 and m/z

into

of the

protocatechuate

170 (molecular 4).

a small

(
as demonstrated

(Fig.

and m/z

152

However,

metabolism

50% of

“02,

was no evidence

incorporated,

No evidence could

observed

at

of methylated

m/z

of

RATIO

4-hydroxybenzoate

4-chlorobenzoate

672

in

the

presence

produce of

of

be found,

139 and 141.

WASS/CHARCE

Figure

50%

at mass units

units by

atom

demonstrated

137,

Peaks

(H012.C6H3.CO+)

labelled

been

3).

approximately

at m/z

with

was formed

2 mass

oxygen

peaks

(3,4-dihydroxybenzoyl protocatechuate

incubated

formed

On analysis,

of paired

incorporation

(Fig.

by peaks

of protocatechuate

to have

was

4-hydroxybenzoate

incorporation

4-hydroxybenzoate. found

4-hydroxybenzoate.

Oxygen

When

amount

ws/cHARcE RATIO of authentic methylated

by enzymic 11Oz.

168,

Vol. 124, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

loo * E 5

we 60,

Iz

40

[Qoj+

[@I]

E z

20 ’ 00

20

I!-,

40

6

. loo.

. I20.

nASS/cHARGE Figure

4

Mass spectrum hydroxylation

of of

methylated 4-hydroxybenzoate

I

.-.. 140

I60

190.

. do

RAT IO

protocatechuate in the

presence

produc of

DISCUSSION An enzyme dehalogenate compound

( 10,

that

as the hydroxyl

this

reaction

than

that

in

the dehalogenation

previously

contrast,

previous

suggested

that

oxygen

reported

the

degradation

of

here

mechanism

related

from

labelled

for

aromatic

as the

for

than

is

the mechanism aliphatic

molecular

of aromatic

donor,

involving

example,

the Milne

and

oxygen.

dehalogenation

for

In have

oxygenases,

responsible

of

rather

by Goldman

by mixed-function

hydroxyl

this

utilizes

one

For

observed

rather

mechanism

metabolism

than that

reported

to glycollate

on the

rather

dehalogenation.

donor

to

oxygen

reaction

We conclude

to that

as a hydroxyl

fortuitous

in

has been

oxygen.

reaction.

closely

studies

molecular

molecular

a hydrolytic

of fluoroacetate

water

step

sp.

dehalogenation/hydroxylation

reported

conversion

first

and reported

and not

indicate

utilized

utilize

this

donor

is more

an Arthrobacter

obtained

results

an oxygenase

enzymic

from as the

The data

11).

indicates

These

(4)

extracted

4-chlo’robenzoate

experiments water

system

which

the reaction

(8,g). The dehalogenating no other

documented

donor.

However,

ring,

catal ysed

protocatechuate,

enzyme aromatic

after

of Arthrobacter hydrbxylases

dehalogenation,

by 4-hydroxybenzoate utilizes

molecular

the

sp. TM-1 which

utilize

second

hydroxylation

3-monooxygenase oxygen 673

may be novel: water

as the

are

hydroxyl

of the aromatic

(EC 1.14.13.2)

as the hydroxyl

there

donor.

to

produce

BIOCHEMICAL

Vol. 124, No. 2, 1984

attempts are being made to further

Currently

and purify

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

characterise

the mechanism

the enzyme responsible for this dehalogenation reaction.

ACKNOWLEDGMENTS The gas-chromatograph/mass

spectrometer

was provided

by the UK Cancer

Research Campaign. We thank Miss Val Bowden for preparing the manuscript. REFERENCES (1857) Can. J. Microbial.,

3, 151-158.

1.

Jensen, H.L.,

2.

Kearney, P.C., Kaufman, D.D. and Beall, Res. Commun.,l4, 29-33.

3.

Motosugi, K., Esaki, N. and Soda, K. (1882) J. Bacterial.

4.

Goldman, P., Milne, 428-434.

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Weightman, Microbial.

A.J., 128,

G.W.A.

M.L.,

and Keister,

Weightman,

A.L. and Slater,

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Klages, U. and Lingens, F. (1981) J. Bacterial.

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Markus, A., Klages, U. and Lingens, Chem., Bd m, 431-437.

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Kaufman, S., (1961). Marks,

J.H.

Chem., m,

(1882)

J. Gen.

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Klages, U. and Lingens, 223.

Brussels,

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D.B., (1968) J. Biol.

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(1964) Biochem. Biophys.

F. (1980) Zbl. Bakt. Hyg. I. Abt. Orig.,

T.S., Smith, A.R.W. and Quirk, 25-29 July.

A.V.

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Marks, T.S., Smith, A.R.W. and Quirk, 48, In press. Microbial.,

12.

Vogel, A.I.,

(1956) Practical

146, 64-68.

F. (1882) Hoppe-Seyler's

Biochim. Biophys. Acta.,

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