Isolation of orsellinic acid synthase

Vol. 32, No. 4, 1968

ISOLATION

OF ORSELLINIC

G. M. Gaucher Chemistry

ReceivedJuly15,

derived

Department, University of Calgary, Calgary, Alberta, Canada.

orsellinic

shown to be derived

according

and M. G. Shepherd

1968

The phenol, metabolites"

ACID SYNTRASE

to the

"acetate

phenols,

acid, from

a linear

hypothesistl

orsellinic

acid

was one of the

(Birch,

first

condensation 1967).

is biogenetically

"secondary of acetate

Of all the

the

units

acetate Only

simplest.

three

FIGURE 1 ORSELLINIC ACID BIOSYNTHESIS

CH+SCoA

cm 0 I II

+

Protein CoASH

1

CHpZ-Protein

WHO I I 3 CH2++rotein

Biosynthetic

3

-

two transacylases + an Acyl Carrier Protein

-

a Condensing Enzyme

16

-

a Cyclization

#7

-

a Hydrolase

#1,2 ry3, 4,

CH+-CHpZ+'rotein 4

II

II

II

CH3-C-CH2-C-CH2-C-frotein 5

- co2

CH3-C~H2X-CH2+Z+ZHpZ-Protein J-1 . N-m@

6

664

Components

5

Enzyme (?) (?)

Vol. 32, No. 4, 1968

BIOCHEMICAL

condensations

are

to orsellinic

acid

hypothetical acid

cell

free

zyme is

fungal

acetyl

extracts

which

first

crude

the

as a source

from the

Commonwealth

minimize

strain

philizea

on current --in vitro reported

in a greater

illustrated

biosynthesis

of or-

the

five

first

synthase.

fraction

than

in the of fatty

acid

a particulate

cyclizes

concepts

for

of orsellinic

An orsellinic

I.M.I.

suspension

with

is

is

which

time

using

This

en-

by differential

fold

purification

over

extract.

madriti,

Mycological aliquots

of p. madriti,

prepared

paper

producing

strain

1962) was obtained

Surrey,

England.

of a detergent from

under

In order

(4 ppm Aerosol

Czapek-Dox

sealed

strips,

of Penicillium

and Gowlland,

Institute,

variation,

on filter

acid

96,506 (Birkinshaw

strain

agar

vacuum

slants,

and stored

to

OT) spore were

lyo-

at approxi-

5O C. Culture

Typical

changes

concentration with

legend

of Figure distilled

were

then

pension

submerged.

conditions

the bulk

with

Conditions

and Preparation

in dry weight, for

culture

42 hours

cultures

and assays

of a 6-liter

with

was sonicated

Sonifier.

for

water cells'

20 minutes

The supernatant

lIn the cold, lyophilized fresh cells are devoid

from

Extract:

grown the

between

acid

illustrated

accompanying

culture,

by packing

distilled

are

in the

by filtration,

Cell-free

and orsellinic

of 2. madriti

fermentor

and dried

of Crude

concentration

detailed

2, was harvested water

slurried

glucose

of 3 g of lyophilized

(pH 7.5) S-110

communication

results

Organism:

mately

based

and malonyl-CoA

chain

This

is

shown to be associated

centrifugation

Cg polyacetate

modification.

1, which

In this from

the

further

Figure

biosynthesis. acid

to yield

without

scheme,

sellinic

the

required

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in Figure legend.

as described

cells

filter

were papers.

and lyophilized

At in the

washed

twice

The cells

overnight.

A sus-

in 80 ml of cold. 0.2 M TES* buffer at the top

setting

a 20 minute

centrifugation

cells retain their activity of activity within 3-4 hours.

*N-tris(Hydroxymethyl)methyl-2-amino-ethane

sulfonic

665

of a Branson

model

(48,000

for

5-10

days,

while

acid

from

Calbiochem.

x g)

2

Vol. 32, No. 4, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GROWTH CHARACTERISTICS

2c

0.7’

14

0.6-

12

% 0.5' 8 - 0.4-

3 :: c!I

2 0.3' E ,y 0.2' d g 0.1'

a B zcl

o-

280 SHAKE

240

CULTURE

642' 0'o

2'

4'

6'

8'

lo

12

14

16

18

20

22 24

280

14 12

26

240

FEEKiWTORCULTURE

4

a

12

16

20

24

28

TIl@

32

36

40

u!

(hours)

Fig. 2. Lyophilized g. madriti samples were regenerated by placing the filter paper strip upside down on a Czapek-Dox agar slant in an 8 dram vial, followed From each regenerated agar by incubation at 28' C for approximately 5 days. vial slant a further 6-1.0 vial slants were prepared by a sterile cotton swab spore transfer, followed by the usual 28O C incubation. A 3 ml detergent spore suspension from one such vial slant was then used to inoculate a 300 ml prescripWhen a uniform green conidial coat had tion bottle slant of Malt Extract Agar. spore suspension formed after 5-7 days incubation at 28O C, a 25 ml detergent prepared from the bottle slant was transferred to a 2-liter erlenmeyer conNaNO 3 , taining 200 mls of the following modified Czapek-Dox liquid medium: 2.0 Q; KH2PO4, 1.0 g; KCl, 0.5 g; MgS04'7H20, 0.5 g; FeS04'7H20, 0.02 g; 40.0 g; Yeast Extract (Difco), 3.0 g, and double ZnS04'7H20, 0.0003 g; Glucose, This shake culture was grown at 28O C on a reciprodistilled water, 1.0 liter. cal shaker (3 cm stroke, 150 displacements/minute) and at 18 hours, a point which corresponds to approximately the mid-point of the linear phase of growth, was used to inoculate a lb-liter New Brunswick Microferm fermentor jar containing 6-liters of the modified Czapek-Dox liquid medium described above. This culture was then grown at 28O C with an aeration rate of 4 liters/minute and a The pH was maintained at 6.0 f 0.1 by the automatic stirring rate of 500 rpm. addition of N NaOH and N HCl using a Beckman combination electrode in conjuncFoaming was controlled by the autotion with a Radiometer automatic titrator. matic addition of small aliquots of Antifoam C (Dow Corning) as necessary. In

666

Vol. 32, No. 4, 1968

handling throughout cultures total

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

all

lyophilized, agar and liquid cultures and all transfers were made in a sterile in particular were checked for bacterial With care being of the cultures

volume

taken to prevent being sampled,

aseptic techniques transfer hood. contamination.

any significant the following

were used Fermentor

change in the culture assays were

used:

1.

Dry Weight - Ssmples were suction filtered through a pre-weighed filter paper .and the mycelial mats were witshed twice with distilled water and dried overnight in an oven at 110' C, cooled to room temperature and weighed.

2.

Glucose - The filtered Biochemical's Glucostat

3.

Orsellinic Acid - To 3 mls of FeC13 reagent (0.04 M FeC13.6H20 in 0.02 M HCl), 3 mls of medium (diluted if necessary) are added, mixed and read in 30 minutes at 540 mn against a reagent blank. The standard plot is linear up to 1.75 li moles of orsellinic acid.

was filtered

through

glass

Enzyme Assay as

in the

detailed

into

orsellinic

2500 apm. we have acid

found yield

wil:L

tures

that

wool

of the

general

susceptibility

of g. madriti activity

which

the

lack

of enzymatic

was added

to the

crude

the

loss

of NADPH resulted other

two peaks.

of both

at the

origin.

replaced

with

the

fatty

buffer

silica

3At O-4' C this after 6 hours,

gel

in the Omission

acid

Controls

chrc'matographic

thesized,

orsellinic

loss

of the

of both

peak at the

the

evidence

that

radiolabeled to the

acetyl

NADPH and

synthesized.

acid

peak

and retention

origin.

orsellinic

in

the peak

was boiled

or

To further

support

acid acid

resulted only

was being

peak from

extract retains approximately 80% of the original while in 24 hours it is devoid of activity.

667

3, if

were

leaving

cul-

and are

acids

enzyme extract

orsellinic

orsellinic

occasionally

in Figure

peaks

crude

variation,

acid

NADPH and acetyl-CoA

the

only

that

orsellinic

fatty

acid

yielded

corresponding

but

average

producing

to the usual

and fatty

assay,

to strain

actively

here,

in addition

and orsellinic

in which

are

As illustrated

acid

standard

of radioactivity

to produce

activity.

enzyme extract

A, both

In the

of fungi

indicated

Worthington

A was on the

which

ability

using

extract".3

incorporation

malonyl-coenzyme

devoid

of the

3, the

labeled

the average

glucose

a "crude

from

therefore

Omission

for

Requirements:

of Figure

cultures

are obtained

malonyl-'coenzyme

to yield

and Cofactor

legend

acid Because

medium was assayed assay.

syn-

a number

activity

Vol. 32, No. 4, 1968

BIOCHEMICAL

THIN

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LAYERRADIOCHROMA~~

300

200

E s! Ef k 8 100

0

Fig. 3. In the standard enzyme assay, the crude extract at pH 7.5 (0.1 ml) was incubated in the following reaction mixture: 2-14C-Malonyl-CoA,' 6.84 x lo4 dpm in 14.8 mu moles; acetyl-CoA,2 10 mu moles; total volume 0.13 ml. After a 10 minute incubation at 21' C the reaction was terminated by addition of 1 ml of N H2SO4 and 0.5 ml of methanol, and extracted with three 4 ml portions of ether. Synthetic orsellinic acid3 (0.25 u moles) was added and the ether extract was evaporated to dryness and redissolved in a minimum of acetone. The entire extract was spotted on a 5 x 20 cm neutral silica gel plate and developed for 14 cm in chloroform-acetic acit (gO:lO, V/V). The plate was then scanned in a Packard Radiochromatoscanner. The silica gel encompassed by the radioactive peak which corresponded to the average Rf of orsellinic acid (0.47) was scraped into a vial containing 10 ml of a scintillator solution5 and counted for 10 minutes in a Chicago nuclear scintillation counter. The incorporation of radioactivity into the orsellinic acid peak ranged from 500 dpm to greater than 10,000 dpm per assay. In the particular assay illustrated by this radiochromatogram NADPH (40 mu moles) was added to the crude enzyme extract in addition to the usual acetyl and malonyl-coenzyme A. 'Prepared

by the

method

2Prepared

by the method

of Trams

and Brady

of Simon and Shemin

668

(1960) (1953).

from

2-14C-Malonic

acid.

Vol. 32, No. 4, 1968

3Prepared

BIOCHEMICAL

according

to Gaucher

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Shepherd

4Apart from scanning for radioactivity detected by the coloration of the spraying the plate with diazotized 5According

of thin

layer

to Snyder

(1964).

plates

was extracted

(200 mg) was added ness. from

to the

The resulting acetic

solid

acid-water.

was 266 dpm per

ether

with extract

crude

in the

supernatant

subsequent natant

of Trautman with

S-rates

a five-fold

after

centrifugation

it

greater purification

extract

(48,000

60 S.

orsellinic

acid

was evaporated

to constant of the

specific

first

to ary-

activity

(113 mg)

crystals

264 dpm

yielded

Orsellinic

at 90,000 x g for the that

acid

synthase

was found x g for

60 minutes enzymatic

this

As indicated

of orsellinic

acid

x g supernatant)

contained

was calculated than

readily or by

recrystallizations

centrifugation

which

acid spot is upon standing

10 mg (13 mg).

at 210,000

a pellet (1963)

and the

Centrifugation:

enzyme extract

yielded

Synthetic

activity

subsequent

per 10 mg (47 mg) and 277 dpm per

in the

ether.

was recrystallized

10 mg, while

press).

the orsellinic spot which occurs p-nitroaniline.

The specific

Differential

(in

to be retained

30 minutes. this

90,000

x g super-

was composed

in Table

1, this

Upon

Using

activity.

pellet

activity

the method

of components

pellet

represented

synthase.

TABLE 1 PRELIMINARY PURIFICATION TOTAL PROTEIN (mg)

FRACTION

SUPERNATANT (90,000 xg; 30 min.) PELLET

(21010

xg; 60 min.)

192 54

INCORPORATION (dpm/assay) 1040

1530

669

SPECIFIC ACTIVITY (dpmlassayl mg protein)

FOLD PURIFICATION

4333

1

22,500

5

Vol. 32, No. 4, 1968

BIOCHEMICAL

Discussion: into

secondary

acid. of the

while

cell-free

the

additional role

CII+ metabolite synthesis

observations

pyrone

derivative,

or its

pigeon

liver

and yeast

et al,

1964;

Lynen,

mediate

whose

carrier

protein

key role

(Money

19671.

components complex"

Also

not

et al,

of the molecular

to fact

is the

of this

In summary it

be necessary in this

of 6-methylsalicyclic at least is

to produce

area.

670

inter-

is

the

desired

an acyl

intermediate 1967;

and

Douglas

and

of the protein

term

"multi-enzyme

latter acid.

that

is

the potentially

some of these

obvious

such as

polyketoacyl

of aggregation

In clarification

that

The non-

Thus

polyketo

of the

suggests

1966).

here

the applicability

weight

in the

hypothesis.

(Bu'Lock,

degree

of NADPH (Brodie,

one of which

Not indicated

mode of cyclization

1966).

complexes.

enzyme bound,

this

by the

metabolites

this

components

systems

synthesized

and Bloch,

supports

The pos-

(3,54iketohexanoic

synthetase

to secondary

in stabilizing

and hence

enzymes will

1965).

indicated

in press)

(Brock

has the

in these

is

acid

1965)

1967).

acid

lactone

fatty

by four

cell-free

acid

in the absence

a growing,

mediated

subsequent

and Hager,

acid

pyrones

of or-

the

1961; Light,

triacetic

1967) further

chelation

(Reed and Cox,

be multi-enzyme

hypothesis

ion

concerned

determination

purified

is

(Vagelos,

its

either

only

and Hermodsson,

intermediate

synthetases

1 indicates

for

1959)

of these

synthesis

6-methylsalicylic

and Tada,

compounds

et al,

synthesis

of metal

specifying

(Light

acid

of appropriate

scheme in Figure

Money,

that

and Mosbach,

as expected

cell-free

(Gatenbeck

triacetic

fatty

Thus

have been reported

1967) and by the E. Coli

conversion acid

the

and malonate

the biosynthesis

acids.

@polyketoacyl

of NADPH and sulfhydryl

orsellinic

for

of NADPH (Lynen

acid)

enzymatic

fatty

of the Cg phenol

by the

(Gatenbeck

between

alternariol

of an enzyme bound

supported

presence

of the

of acetate

acid

similarity

requirements

requirement

tulated

incorporation

are required

Identical

synthesis

in

close

and malonyl-CoA

sellinic

the

the

and the biosynthesis

acetyl-CoA

cell

such as orsellinic

supported

metabolites

is

The whole

metabolites

has uniformly

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

further transition

point,

a recent

synthetase systems work

may with

from

Vol. 32, No. 4, 1968

BIOCHEMICAL

Acknowledgements: National

Research

of Research

Council

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This

work

of Canada

was supported

and by a grant

by Grant

from the

A-3588 of the

Brown-Hazen

fund

Corporation. References

Birch, A. J., Science, 156, 202 (1967). Birkinshaw, J. H., and Gowlland, A., Biochem. J., 84, 342 (1962). Brock, D. J. H., and Bloch, R., Biochem. Biophys. Res. Comm., 23, 775 (1966). Brodie, J. D., Wasson, G., and Porter, J. W., J. Biol. Chem., m, 1346 (1964). Bu'Lock, J. D., Essays in Biosynthesis and Microbial Development, Wiley, New York, 1967, p. 32. Douglas, J. L. and Money, T., Tetrahedron, 3, 3545 (1967). K., Acta Chem. Scan&., 13, 1561 (1959). Gatenbeck, S., and Mosbach, Gatenbeck,, S., and Hermodsson, S., Acta Chem. Scand., lp, 65 (1965). Gaucher, G. M., and Shepherd, M. G., in Law, J. H. (ea.), Biochemical Preparations, Vol. 13, Wiley, New York, in press. Light, R. J., J. Biol. Chem., 242, 1880 (196-f). Light, R. J. and Hager, L. P., Arch. Biochem. Biophys., in press. Lynen, F., and Tada, M., Angew. Chem., 73, 513 (1961). Lynen, F., Symp. on Organizational Biosynthesis, Rutgers, 1966, Academic Press, New York, 1967, p. 243. Money, T., Comer, F. S., Webster, G. R. B., Wright, I. G., and Scott, A. I., Tetrahedron, 3, 3435 (1967). Reed, L. cJ., and Cox, D. J., Ann. Rev. Biochem., 3, 5'7 (1966). Simon, E. J., and Shemin, D., J. Am. Chem. Sot., 75, 2520 (1953). Snyder, F., Anal. Biochem., 2, 183 (1964). Trams, E. G., and. Brady, R. O., J. Am. Chem. Sot., 82, 2972 (1960). Trautman, R., in Williams, J. W. (ea.), UltracentrifuRal Analysis, Academic Press, New York, 1963, p. 204. Vagelos, :P. R., Alberts, A. W., and Majerus, P. W., Ann. N.Y. Acad. Sci., 131, 177 (1965).

671