Anthranilic acid hydroxylase from Aspergillus niger

Vol. 31, No. 2, 1968

BIOCHEMICAL

AZl?I;RANILIC P. V. Subba Rao, Department

Received

March

and

paspali Subba

conversion

acid

was shown

and his

al.

by Groeger (1967a)

niger ring-labeled)

anthranilic been

so far

is

from

catechol.

different

microbes

from

the

and higher

of

Claviceps

More

recently, of 14

D&tryptophan-C

3-hydroxyanthranilic

However, acid

the

paper

acid

we report

acid

of

preparations

hydroxylase

anthranilic

acid,

conversion

by cell-free

present

anthranilic

anthranilic

felts

from

acid,

In the

a soluble

mycelial

radioactivity

to 2,3-dihydroxybenzoic

of

which

and

to

(19651.

washed

anthranilic

demonstrated.

demonstration niger

C. S. Vaidyanathan of Science,

cultures

co-workers

that the

into acid

acid

not

NIGER

tryptophan

by submerged

reported

incorporate

2,3-dihydroxybenzoic

has

of radioactive

2,3-dihydroxybenzoic

Aspergillus

FROM ASPBRGILLUS

N. S. Sreeleela, R. Premkumar and of Biochemistry, Indian Institute Bangalore-12, India

vivo

Rao --et

(benzene

ACID HYDROXYLASE

12, 1968

The --in acid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

from

Aspergillus

hydroxylases

reported

plants. EXPERIMER'I'AL

Aspergillus flasks

niger

on a synthetic

anthranilic

The

washed

Washed glass

medium

acid.

sporulation,

powder

containing

0.001

the

extract

(27

ml)

mycelia

(IO

et

were with

M GSH (30

ml).

After

at

IOOOOxg

stirring,

0.025

passing

2% protamine

193

at

at

30'

in

supplemented

water

M sodium

for

hr.

before

distilled

with

48

1956)

harvested

extracted

with

for

al.,

g> was macerated

was centrifuged

was added

was grown

(Byrde

repeatedly

mycelium and

(UBC 814)

the

with

advent

and stored

ho with

equal

1 litre O.l?A

of at

-20

weight

of

phosphate

buffer,

pH 7

through

a cheese

cloth,

'lo min. sulfate

To the solution

0

supernatant (3 ml>

.

Vol. 31, No. 2, 1968

and

the

of the

BIOCHEMICAL

resulting

precipitate

supernatant

which

has

been buffer

suspension

was

layer

were

et al.,

(400

used

10

or

to

to

ethyl

acetate

paper

using

ponding

to

compound

from

and

0.5

0.025

30 min.

and the

>I

the

clear

filtrate

at

of

and

2% formic

ml of

shaking

RESULTS

from

anthranilic

acid

acid

hydroxylase

was

(Kair the

mixture

extracted

the

ether

Vaidyanathan,

isolation

1964).

of

the

incubation

mixture

anthranilic

acid,

incubated

for

1 hr.

at

twice

with

equal

volume

anhydrous

chromatography

Na2S04,

residue

The eluted

hot

No.

fluorescent with

the

30°,

hot

1

of

solvent

was dissolved

on Whatman

solvent. was

and

(Venkataraman

25 mg of

in

filter

band

corres-

95% ethanol.

The

water.

AND DISCUSSION

of

the

reaction

product:

in

the

presence

of

identified

and

unit) was

from acid

The

from

the

buffer,

with

acid

and

aliquots

temperature.

as the

reaction

scale

extracted

crystallized

Identification

ml)

(0.2

a large

and

to

The

For

were

2,3-dihydroxybenzoic finally

glucose-6-

(0.2

enzyme

N HCl

acid

umole),

anthranilic

acid,

100

subjected

NADP (0.4

30'.

product:

ml of Tris-HCl

room

pH 8.2

acid

100

at

buffer,

Suitable

anthranilic

1

Tris-IX1

dehydrogenase

N HCl

reaction

After

dryness

was

ml

DEAE-cellulose

pH 7 with

funnel

umole),

2,3-dihydroxybenzoic

ether.

was taken

Fifteen

of

After

containing

estimation

pH 2 with

peroxide-free

1962).

ether.

of the

NADPII,

acidified

Sober,

20 min.

of

the

containing

mg of

to

(0.4

for

addition

for

product

ml)

adjusted

glucose-6-phosphate

incubated

Isolation enzymic

and

a Buchner

acid

peroxide-free

1948)

weight)

(2 ml)

umole),

by the

5 ml of

2 g (wet

through

mixture

was

terminated

by centrifugation.

enzyme.

(0.5

1 ml enzyme

with

and

anthranilic

umoles),

with

washed

filtered

as the

phosphate

stirred

(Peterson

A reaction (60

was removed

previously

phosphate

was used

was

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

partially

as 2,3-dihydroxybenzoic

194

The

enzymic purified

product

formed

anthranilic acid

by comparing

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 31, No. 2, 1968

its

properties

the

compound

acid

in

with

solvents

an authentic

(Reio,

and Towers,

1960).

ethanol

was

identical

showing

peaks

at

with

248 and

Incubation

of

that

to

with (Ibrahim

isolated

product

2,3-dihydroxybenzoic

isolated

carboxy-lyase

catechol

decarboxylation

chloride

the

enzymic

reactions

ferric of

authentic

and

color

and

spectrum

enzymically

acid

decarboxylation

similar

acid

of

synthetic

in

acid

316 mu.

the

2,3-dihydroxybenzoic

gave

ultraviolet

2,3-dihydroxybenzoic

30th

sulfanilic

The

Chromatographically,

synthetic

1938).

acid

diazotized

sample.

from

of 2,3-dihydroxybenzoic

I-nitroaniline,

the

of

was indistinguishable

different

samples

those

(Subba

product

product

from

A.

Rao et al.,

was established

with

the

niger,

resulted

196713).

The

by comparing

purified in

its

identity

of

with

authentic

acid

hydroxylase

catechol. Optimum

pH and

had an optimum Various

alone

like

or

further

course

in the

to mole

the

data

and

an absolute l%N and

combinations. the

addition

requirement FAD could Even

of

stoichiometry:

presented

Even

though

acid,

2,3-dihydroxybencoic

in Table

I,

it

acid

the

formed

in

the

crude

apparent

anthranilic

crude

XADPH.

replace preparations,

preparations

treatment

of

is

in

not

for

KADPH.

removal

between

acid

acid presence

that

with

DEAR-cellulose

acid

carboxy-lyase.

there

was a mole

disappeared of

and

partially

purified

acid

hydroxylase

hydroxylase.

Taniuchi

et al.

Pseudomonas

which

catechol

showed

KADH, THFA,

various

complete

2,3-dihydroxybenzoic

to

and

Anthranilic

2,3-dihydroxybenzoic

relationship

anthranilic

requirement:

without

metabolized

resulted

from

in

was no activity Time

8.2

cofactors

NADPH either

Prom

pH around

other

there

cofactor

without

(1964) catalyzed

apparently

reported

an anthranilic

the

direct

involving

195

conversion any

detectable

of

anthranilic

intermediate.

acid

BIOCHEMICAL

Vol. 31, No. 2, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLZ Time Time

in

I and

course

Anthranilic disappeared

min.

stoichiometry acid

2,3-Dihydroxybenzoic acid formed (umole)

(umole)

15

0.036 0.054 0.065

0.033 0.052 0.065

go”

0.094 0.145

0.090 0.138

5 10

This

enzyme

showed

hydroxylase oxygenase acid

in

Partially a third acid

from which the

purification

Tecoma

purified

of

in

involved for

hand,

acid

NADPIi and TIIFA

is

properties

other

anthranilic

acid

a requirement

NADH and FAD.

on the

anthranilic

which

and

for

stans,

hydroxylates

presence

type with

a requirement

(Nair

a simple

and Vaidyanathan, from

formation

of

Further

of

are

enzyme

acid nono-

to 3-hydroxyanthranilic

RADPH alone. this

is

hydroxylase the

Anthranilic

in

A.

1965).

niger

represents

2,3-dihydroxybenzoic studies

on the

progress.

We wish to thank Dr. R.J. Bandoni, Department of Botany, of British Columbia, Vancouver, Canada for providing the culture One of us (P.V.S.) is Prof. P.S. Sarma for his keen interest. Scientists' Pool Officer.

University and a C.S.I.R.

REFEREiiCES tiyrde, R.J.W., Harris, J.F. and Woodcock, D., Biochem. J., 2, 154 (1956). Groeger, D., Erge, D. and Floss, R.G., Z. Naturforsch., E, 856 (1965). lbrahim, R.K. and Towers, G.H.X., Arch. Biochem. Biophys., 3, 1.25 (1960). ijair, P.M. and Vaidyanathan, C.S., Anal. Biochem., 7, 315 (1964). Nair, P.;4. and Vaidyanathan, C.S., Biochim. Biophys. Acta, 110, 521 (1965). Peterson, %.A. and Sober, H-A., in Methods in Enzymology, Vol. 5, edited by Colowick, S.P. and Kaplan, X.0., Academic Press, New York, p-3 (1962). Reio, L., J. Chromatography, 1, 338 (1958). Subba Rao, P-V., I;oore, I;. and Towers, G.H.N., Biochem. Biophys. Res. Commun., 23, 1008 (1967a). Subba Rao, P.V., $1oore, 1;. and Towers, G.H.N., Arch. Biochem. Biophys. 122, 466 (1967b). Taxchi, Ii., Hatanaka, PI., Kuno, S., Hayaishi, O., Nakazima, I4., and VeI~~~f~r;~~~.~.~-V~~~~~a~~~~~~ 173, 641 (1948).

%!I

~~~",~~?~~',.B.,

196

J.

Biol.

Chem.,