A catecholic 9,10-seco steroid as a product of aerobic catabolism of cholic acid by a Pseudomonas sp.

A CATECHOLIC 9,10-SEC0 STEROID AS A PRODUCT OF AEROBIC CATABOLISM OF CHOLIC ACID BY A PSEUDOMONAS SP. Robert

J. Park,a*

Noel W. Dunn,b and John A. Ideb

“CSIRO Division of Food Research, Meat Research Laboratory, P.O. Box 12, Cannon Hill, Queensland 4170, Australia b University of New South Wales, Department of Biotechnology, Kensington, New South Wales 2033, Australia Received November 23, 1986 Revised March 9, 1987 ABSTRACT A mutant of the efficient bile acid-utilizing Pseudomonas ptitida ATCC 31752 was found to accumulate three major catabolites on aerobic growth on cholic acid. One of these catabolites was isolated and identified as 3,4,7,128-tetrahydroxy-9,lO-seco-l,3,5(1O)-androstatrieneThis Is the first catecholic 9,10-secosteroid isolated 9,17-dione (2). from the micrcbial degradation of bile acids or sterols and confirms the role of such secosteroids in the microbial degradative pathway for steroids. INTRODUCTION Study

of

natural

bile

number

of

acids

reports

(l-8,

11,

of

bile

degradation (8,

10).

degradation

of

in the early

steps.

As part bile

acid

of

mutant

isolated

(14).

plates

containing

STEROIDS

bile

12)

degradation species

and a partial

acids

or catabolism

has

been

Var ions

(l-12).

This

pathway

differs

acids

by gram-positive

the

of various

subject

cataboli

catabolic

by gram-negative

a program to produce

catabolites

indticed

microbial

by Pseudomonas

recent

identified

proposed

the aerobic

tes

pathway pseudomonads

from

that

organisms

high yields

of

(131,

This

of

produced cholic

48 / 5-6

an auxotroph

of

a water-soluble

acid

as a source

November-December

for

the

has

been for

valuable (MNNG)-

P.putida

carbon.

been

particularly

potentially

ATCC 31752

red pigment of

a

have

proposed

an N-methyl-NV-nitro-N-nitrosoguanidine

(PS5-7)

of

was

when grown

In this

1986 (439-450)

report

on we

439

440

Park et al

examine

the

fermentor

major

products

and demonstrate

of

catabolism

the

accsmGlation

of cholic of

acid

a catecholic

by PS5-7 in a 9,

IO -

secosteroid. EXPERIMENTAL UV spectra were recorded in MeOH on a Hewlett Packard 8450A spectrophotometer, PMR spectra in CDCl3-d6-DMSO, on a JEOL JNM-PS-100 spectrometer at 100 MHz, with TMS as internal standard. Low resolution mass spectra were obtained at 70 eV on a Varian MAT 311A instrument with a Spectra-system 100 data system. High resolution mass spectra were recorded on the same instrument. Melting points were uncorrected. HPLC analyses were performed with a Waters M45 pump connected to, in series, a Waters U6K injector, a micro Bondapak Cl 8 column, a Varian Varichrom UV-visible detector, and a Waters R401 Differential Refractometer. For the analyses of cholic acid utilization and product formation, the culture samples were processed through a Waters Sep-pak ~18 cartridge, and analyzed by HPLC. The methanol extract obtained from the Sep-pak processing was injected (10 vL) using 75% aqueous methanol containing 0.2% acetic acid as solvent, at a flow rate of 0.7 mL/min to measure cholic acid utilization. For the analysis of product formation the sample (10 uL) was injected using 40% aqueous methanol with 0.2% acetic acid as solvent at 0.7 mL/min. Under these conditions ca taboli tes _’ 1 _) 2 and -3 eluted at 24, 18.5, and 21.5 min, respectively. The microorganism PS5-7 was an MNNG-induced mutant of a cryptophan auxotroph of P.putida ATCC 31752. PS5-7 was affected in the bile acid catabolic pathway ( lha). It was maintained on Nutrient agar slants; PS5-7 was grown either in shake flasks (9) or in fermentors as previously described for the parent strain (lo), during study of the formation of steroid catabolites from bile acids. Inoculum preparation, growth conditions and medium, meastirement of W-absorbing products, and TLC monitoring of substrate utilization and product formation were performed as described before (9-11 ). The catabolites were recovered from the fermentor culture after centrifugation, absorption on a styrene-divinylbenzene-copolymer column, subsequent elution with methanol and evaporation, as described before (14a). A portion (3 g) of the solid product (5.6 g) was dissolved in dichloromethane (ca. 20 mL) and chromatographed on a column containing silica gel (150 gr(Merck, 230-400 mesh, Art. No. 9385), and the individual components were eluted with dichloromethane containing from 4 to 10% v/v of ethanol. Fraction 1 (0.20 g) eluted with 4% ethanol, fraction 2 (0.48 g) with 6% ethanol,and fraction 3 (0.92 g) with 8% ethanol. 3,7,12g-Trihydroxy-9,10-seco-1 Fraction yielded nearly

3 above, colorless

,3,5(10)-androstatriene-9,17-dione recrystallized needles (0.21

(1) -

twice from aqueous methanol, g) mp 158-1590, melting at

CATECHOLIC CATABOLITE OF CHOLIC ACID

158.

on

5-159.5O

3,7,12g-trihydroxy-9,10-seco-1 (MeOH) 217,

217 241,

E 4.39)

(log10

and

300

admixture with ,3,5(10)-androstatriene-9,17-dione; and 280 nm (log10 E 4.00) and

44 1

authentic h max h max (MeOH+OH-1

run.

3,4,7,12~-Tetrahydroxy-9,10-seco-1,3,5~10~-androstatriene-9,17-dione Fraction

2

above,

chloroform-methanol h max

176-177"; h max

(MeOH+OH)

1628, J

1594,

= 8 Hz,

and

H24

217

I-H),

provided

216

(log10

and

290

nm;

1461,

817

cm-';

6.46

2.23

06

recrystallized

v/v),

(MeOH)

1472,

12a-H),

(Cl9

(9:l

(IH,

(3H,

requires

s,

d,

Catabolite

3.93)

E

IR

max

J

= 8 Hz, 1.12 19-CH31,

348.1573);

m/z

and

(KBr)

6 (in

2-H), (3H,

times (0.62

283

with

nm

S,

from

3310,

E 3.36);

1732, 6.57

1702, (IH,

(2H, complex MW 348.1579

peak),

m/z

348

m/z

CM+-137-H20),

182

mu

g),

(log10

DMSO-d6)

3.9-4.20 I8-CH3);

(base

193

three needles 3430,

CDCl3

137

m/z 211 CM+-137), m/z CM+-H20), 166 CM+-182), m/z 148 CM+-182-H20).

330 M/z

colorless

(2) -

d, 7-H

CM+),

m/z

CM+-166),

3

Fraction 1 from the silica gel chromatography above prodticed a red-brown oil (0.22 g) on evaporation. This oil was dissolved in methanol (5 mL) and chromatographed on a column containing Lichrosorb RP-8 (60 g) (Merck Art. No. 9324). The components were eltited with 50% v/v aqueous methanol. The principal fraction was evaporated to give a nearly colorless oil (0.18 g;), which deepened to a red-brown color on standing. This oil was dissolved in dichloromethane and again chromatographed on a silica gel column (50 g) (Merck, 230-400 mesh, Art. The main component el>uted with dichloromethane containing No. 9385). 2-4s methanol to give a red-brown oil (0.15 g) on evaporation. Examination of this oil by HPLC analysis showed it to contain ca. 94% of one component eluting at 21 .5 min with 40% v/v aqueous methanol containing 0.2% acetic acid as solvent. This oil showed X max (MeOH+OH-) 298, 238, and 219 nm. RESULTS

AND

DISCUSSION

Small-scale were

grown

with

20

added, this 48

on

mg/L

and

their

way,

it

h of

shake 2g/L of

sodiLun

L-tryptophan.

was

shown and

showed

absorbance

develop

in

cultures

acetate

catabolism

addition

the

flask

culture,

as

that three

cholic main

MNNG-induced source

bile

acid

salts

by HPLC and acid

prodticts

was

of

completely

were

carbon

A red

color

deepened

to

a bright

red

PS5-7

together were

then

procedures.

In

consumed

formed,

nm.

mutant

(2g/L)

TLC

280

near

which

the

a primary

Variotis was followed

maxima

of

all was

within

of

which

observed

48 h after

cholic

to

442

Park et a/

acid

addition

and

turned

to a red-brown

development

had been noted

ATCC 31752

in

severely salts

the

limited of

sodium

tatirocholic

maximtim was less

one

from

catabolite

from

of

the

to two or nm.

appropriate

earlier,

bile

acid

was then detail

carried

and

to

out,

of

of

to

provide

identification formation

growth

products.

was monitored

The PS5-7

source

of

at

(2g/L)

was added.

and in relative (as it

measured

After

while

all the

of

the

catabolite

products,

bile

indicated

that

probably

the

the

catabolites

presence

the catabolism material

of had

growth

in

the

(10).

of to

allow

Catabolism

of

cholic

the bile

of

acid

acid

in more

separation

and

cholic

and

the

acid

by the HPLC, spec tropho tome tr ic, and on 2 g/L

incubation

glycerol

for

acid

and the

as an initial

21 h sodium

Changes in the 280 nm absorbance

by HPLC procedures) that

in

was grown

amounts of cholic

can be seen

addition,

25OC.

and

and dihydroxy

ATCC 31752

aeration

PS5-7

study

TLC procedures. carbon

P.putida

sufficient

the

products

of

the

derivative

under restricted

A fermentor-scale

manner,

color

These

following

the

the

was

hydroxylated

isolated

sodium

prodticed

However,

acids

9,10-seco-1,3,5(10)-androstatriene-9,17-dione. been

under

the

three

HPLC and TLC data bile

P.putida

hyodeoxycholic,

monohydroxy

to orange.

each

corresponding

these

of

an analogous

280

color

took place

with PS5-7 also In

near

parent

The use

(10).

catabolized

with

yellow

the

A similar

when growth

acids

absorbance

intense

of

chenodeoxycholic,

were each fully

varying

acids,

acid,

concentrations

deoxycholic,

acids

development

cholic

and glycocholic

of

showing

of

72 h.

growths

and the red coloration.

salts

lithocholic all

presence oxygen

same catabolites

in fermentor

after

three

of

fermentor

major

1,

acid

1.

was consumed

identified

as

samples

catabolites

are summarized in Table cholic

cholate

the

From this 26 h after expected

CATECHOLIC CATABOLITE OF CHOLIC ACID

3,7,12-trihydroxy at

this

hand,

time, was

as

was the

increasing The

terminated. is

phenolic

attributed

in

slight to

the

9,10-secosteroiq

catabollte

The

2.

concentration decrease

in

1,

up to total

decreasing

was

at

its

maximum value

2,

on

catabolite the

time

absorbance

that of

concentration

443

280 of

the

the

other

growth

was

nm after the

4 h

phenolic

secosteroid.

HO

TABLE

1

Changes in the relative amounts’ of stibstrate and major catabolites and absorbance at 280 nm of aliquots from the fermentor catabolism of cholic acid by mutant PS5-7

Cataboll Time

(h)

0.D.280

Substrate

23 28

.02 .02

100 81

33 47 s3

.07 .25 .22

66 22 0

l

Calculated response at 23 h.

of

from the ratio of the HPLC refractive that component from any Lime to that

tes

1

2

3

0 0

0 0

0 0

16 24 19

11 43 36

index detector of the substrate

: 20

444

Park et al

From these were formed

data,

Amberli

evaporation

te

obtained

were recovered XAD-2,

(14b).

chromatography

on silica

by similar

secosteroid

with

exhibited

absorption

a yellow

identified

al

at

was

two of The

peaks these

remaining

for

m/z

valges

of

were also

found

in

peaks

showed

137, the

with

gave

138,

This

the

chat

specsrtin

cataboli

groups

te

soliition,

the formation

presence

of

reported

a

for

synthesized

evidence

for

the presence

and an aromatic

oxygen

specirzm 166,

phenolic

an increase

an additional 1.

Oby

mass

ion with

to

mass

presence

shown in Fig.

9,l

of

ring,

the 3,4-ciihydroxy-9,10-secosteroid

peaks from the phenolic

fragmentation

resoli;tion

hydroxide

carbonyl

corresponding of

of

The low resolution at

by -3 were

phenolic

,3,5(10)-androstatriene-9,17-dione,

to that reported

intense

the

ClgH2406.

similar

again

(15).

separated

in catabolite

as

consistent

and

and cyclohexanone

Sih -et al

and subseqijent

and 283 nm in methanol

on the addition

cyclopentanone similar

216

of

The IF? spectrum

(15).

then

rich

a high

formtila

maxima

3,4-dihydroxy-9,10-seco-1

macrorecictilar

(12).

2 QrodLCed

system

et --

specimen

This

on the

,3,5(10)-androstairiene-9,17-dione,

a molecular

color.

catecholic

fractions

-1 was readily

were destroyed

-1.

2 were

separation.

catabolite

consistent

by Sih

_1 and

te -3 and probably

with methanol

ehromatographic

with an atithentic

The psi-e

secosteroid

while

gel,

cataboli

by elzition

3,7,12B-trihydroxy-1

comparison

of

followed

that

by adsorption

The catabolites

The catabolite

which

was concli;ded

to the phenolic

sl;bsequent

The catabolites resin

it

of

t82,and

a pattern

193.

9,10-secosteroid 16 in

seeosteroid, in

showed

those

The PMR spectrum

m/z

value

of

of

The latter l(12). over

the

with

the

and with

the

consistent fragments

of

catabolite

-2 also

CATECHOLIC

showed

similarities

was described multiplet and and

by at

12

major

catabolites

1 _’

at

Hz)

6.57

(J

at

6.56

exhibited The

This

The attr

to

other

at lower ibtited

and 6.56 at

fine

us

to

proton

non-aromatic

methyl

the

to

at

the

1 .12

445

spectrum

unresolved

protons

and

protons

to

at

2.23

at

(c

C 18

C 7 1.07

and

19

Of

was

two

aromatic

coupling

to

the

three to

protons

C 1,

resonance

at

compared

the

to

three

protons, quartet at

C 2 and

C 6)

was

two

resonances

centered

pattern

these

proton

aromatic

resonance

the

of

J = 8 Hz),

assigned

broadened

spectra

the

(J

= 8,

6.68 to

found

2

(which C 4 (12).

in

related

(12). in the

of

the

the

C

in

of

two

C 4 in

_2.

coupling

with

resonance

in group

and

appear

similarity

the

phenolic

the

at

these

a hydroxyl

The

identical of

a proton

1 proton

catechol

of

of

vicinal

influence

resonances

portions

of

only

(16).

an

PMR spectra

absence

the

protons

with

PpFi

only

ppm.

proton

the

doublets,

the

C 2 exhibited shift

of

6.93

C 2,and

respectively

consistent

assigned

the

whose

included

singlets

between

ppm (both

aromatic

corresponding ppm,

in l),

to

Fig.21,

-1 (see These

three-proton

6.46

difference by

proton

4.04

presence

secosteroids

ascribed

(c-f

assigned

the

= 8 Hz)

three

phenolic

in

6.68

only

same

L),

and

6.93,

dotiblet

catabolite (12).

difference

was

at

ppm, with

in

of

ACID

(12).

The

in

that

previously

6 4.06

ppm

respectively

2,

us

together

(a),

2.20

in

to

CATABOLITE OF CHOLIC

phenol of and

substitution respective

the

and

structures.

was

Accordingly, the

proton

the on C 1.

catabolite at

6 values

catecholic

catabolites

C 4,

at

6.82 of

-2 since

the

and

these

7.09 and

secosteroids

stereochemistry

is

of is

in

the

Park et al

446

HO

.

l.

a

2

Probable mass spectral fragmentation catecholic 9,10-secosteroid -2.

FIGURE 1 :

This

evidence

is

consistent

tetrahydroxy-9,10-seco-1 stereochemistry with

that

stereochemistry of

for

Following cataboli

te

for at

~8 and Cl2 positions the

phenolic

the asymmetric

this

catabolite

altered

the cholic reverse

acid

to give behavior

C7 remains

and normal

maxima at 298,238, to

that

from HPLC and TLC retention

be less

polar

red spot

on spraying

steroidal

either with

catabolites

2.

The

while, unassigned.

likewise,

the

The full

name

phase

2. chromatographic

Examination

by UV spectroscopy

similar

3,4.7,12-

asstimed to be identical

secosteroid

catabolite

to crystallize.

than

structure

,3,5(10)-androstatriene-9,17-dione

However,

the

is

the

for

3 was seen by TLC and HPLC to be almost

not be induced

ion,

the

3,4,7<,12B-tetrahydroxy-9,10-seco-1

is proposed

the proposed

for

,3,5(10)-androstatriene-9,17-dione

at

proposed

with

points

of

purification,

pure (94%),

the

purest

but could

fraction

of

showed maxima at 284 and 210 nm which and 219 nm on addition of

the

data

catabolites the anisaldehyde on TLC plates.

phenolic te

color

1(15).

3 was adjudged

Catabolite

reagent, This

hydroxide

secosteroid

cataboli -1 or -2.

of

used

to

2 produced to

a

visualize

was identical

with

447

CATECHOLK CATABOLITE OF CHOLIC ACID

i

I

I

I

I

I

I

I

7

6

5

4

3

2

I

FIGURE 2:

100 MH, Proton resonance spectra of Cl) 3,7,12$-trihydroxy9,10-seco-1,3,5(10)-androstatriene-9,17-dione and (2) 3,4,7,12~-tetrahydroxy-9,lO-a@co-l,3,5(10)-androstatriene9,17-dione.

448

Park et a/

that

from

strong

catabol

blue

cataboli

1,

color.

while

No further

catabolite

secosteroid

be

secosteroid

skeleton

to

be

The pathways

acids

(8,lO)

variously

androstatriene,

such

made

-1 produced

to

is

4-hydroxylation

such

in

the

cholic

a

characterize

of

the

steps

as

catabolites analogy

they

apply

subsequent with

the

the

degradation

retro-aldol

the

of

acid

phenolic

sterols of

by a spontaneous

well

as

the steroid

non-enzymatic

-1.

known meta

compound, aromatic

cleavage

only

other

the

The next step

pathway.

are shown in Figure

shown for

of

,3,5(10)-

then undergoes

II and _5 are

schemes

(13)

rearrangement

catabolite

-2, which

acids

such

degradative

of

isolated

a 9,10-seco-3,4-dihydroxy

to bile

to 2,

sterols

the catabolism

catabolite via -

or

9,10-

a 3-hydroxy-9,10-seco-1

to give

A ring

catecholic

acids

postulate

to

acid

first

catabolite

for

as in the cholic

proposed

the bile

controlled)

intermediates

These

of

followed

’ thermodynamically

degradation

be

proposed

1,4-diene-3-one

3,

in which

postulated

steroids

(13,

by 15,

18). When this

catecholic

3,

catabolites role

study

has

catabolism the

pathway was sttidied II- dihydroxy

were never

their

in

secosteroid were

a further

by 9a_hydroxylation,

as

to

from catabolism

assumed

1.

and bile

17,

phenolic

attempts

-2 appears

resulting

must

(i-*.e

the

te 3.

The

and

ite

-

isolated

by earlier

9,10-seco-1

from

indirect

demonstrated

that

the catechol

acid

degradative

of

bile

(15,

or bile

of

17-20),

this

and catabolism

acids,

but

The present

17).

2 is a product

the role

(15,

androstatriene

sterols

evidence

and affirms

pathway

,3,5(10)-

from degraded

was implied

of a bile

investigators

of class

the microbial of

compound

by Pseudomonas

microorganisms. The

identity

of

the

cholic

acid

catabolite

-3 is obscure,

but the

CATECHOLIC CATABOLITE OF CHOLIC ACID

0 ir

CCD, +

FIGURE

3:

Ha0

I

449

COO”

I

r

I

co1 + HI0

Abbreviated pathway for the aerobic degradation of bile acids by Pseudomonas spp., including for the formation of phenolic (1) and catecholic (2) 9, 1 0-secosteroids and their subsequent degradation. Cholic acid is the bile acid depicted here.

450

Park et al

evidence

presented here indicates that it may be a product of the

further catabolism of the catechol 1, such as the di-seco compound 4 (Fig.31 or a further catabolite.

ACKNOWLEDGMENTS This investigation was funded in part by a special grant from CSIRO and, in part, by the Australian Meat and Livestock Research and Development Corporation. We thank Mr. I. Criffithsof the Meat Research Laboratory for technicalassistance. The mass spectra were recorded by Mr. K. Shaw of the Food Research Laboratory of the Division of Food Research of CSIRO and the PMR spectra by Miss L.K. Lambert of the University of Queensland, through the courtesy of Prof. C.J. Hawkins of that institution. NOTES AND REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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