A cobalt-free corrinoid compound in Streptomyces olivaceus

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 39, No. 1,197O

A COBALT-FREE CORRINOID COMPOUNDIN STREPTOMYCES OLIVACEUS Kazuyoshi Laboratory Chemistry,

Sato,

Shoichi

Shimizu

and Saburo Fukui

of Industrial Biochemistry, Department Faculty of Engineering, Kyoto University,

of

Industrial Kyoto, Japan

Received February 26, 1970 SUMMARY

An unknown orange-red pigment was obtained from the cells of Streptomyces oliv&eus 6b5-grown on a cobalt-free medium. This comnound showed similar behaviors to those of the cobaltfree corrinoids of Chromatium isolated by Toohey in W, CD-and fluorescence spectra. However it exhibited a more acidic character in paper electrophoresis. Although an attempted insertion of cobalt atom has been unsuccessful , available evidence indicates that this pigment is the first cobalt-free corrinoid found in nonphotosynthetic bacteria, having carboxylic acid side-chains on its corrin nucleus. Corrinoid

compounds containing

some photosynthetic

no cobalt

in

Despite widely,

the fact that this stimulating work has aroused interest such kinds of compounds have not been obtained from non-

cells of deficient

by Toohey

(1)

dis-

covered

photosynthetic bacteria. scribe the isolation of

bacteria

were first (2).

In this communication we wish to dea new cobalt-free corrinoid from the

a strain of Streptomyces conditions.

olivaceus

grown under

cobalt-

MATERIALS AND METHODS Streptomyces

olivaceus

605 used in this

study

was supplied

from Kyowa Hakko Kogyo CO., Tokyo, Japan, This organism has been known to produce a large amount of corrinoids, both complete and incomplete forms (3), in cobalt-sufficient media. For the present study, this organism was cultivated in the same glycerol medium as mentioned in our previous paper (3) except that CoC12 The cells were harvested at the middle exponential was omitted. phase of growth. The isolation procedure of the pigment from the cells

was similar to those of ordinary carrinoids reported earextraction , purification by the usual phenol (4) : Ethanol

lier treatment

and P-cellulose

column chromatography, 170

The same pig-

Vol. 39, No. 1, 1970

ment was also

BIOCHEMICAL

obtained

from

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a protein

fraction

(NH ) SO -precipitate ) of cell-free 42 4 method had an advantage in decreasing

extracts. other

( 0 to 70 % Since the latter contaminating pig-

ments, the following procedure was employed : The wet cells, pended in the same amount of KP04 buffer ( 0.1 M, pH 7.5 ), sonicated trifuged solid

for 10 min at 20 KC. The resulting homogenate was cenat 13,000 X g for 30 min. To the supernatant was added

(NH4j2S04

ration

suswere

and the fraction

was resuspended

precipitating

in ethanol

to

70 % of

( 20 ml per g of protein

satu)-

This

suspension was boiled for 20 min and centrifuged at 13,000 The supernatant was concentrated to dryness -in X g for 30 min. vacua and the resulting residue was, after dissolved in a small amount of distilled ment as described obtained

water , purified by the usual phenol treatin our previous report (4). The pigment thus

was further

cellulose

equilibrated

purified

in the paper

at various

per cm using Toyo Lyophillization pH.

orange-red

powder having

electrophoresis

two-fold

and methionine was not formed

Absorption

fluorescence.

and

Jasco ORD/UV/CD-5 spectrophotometer. was carried out using Hitachi spec-

MPF-2.

RESULTS The yield of the pigment was -ca. 25 pg per grown in the cobalt-deficient glycerol medium. about

performed

filter paper No, 50 ( 20 X 400 mm) of the eluate gave an amorphous

a reddish

CD spectra were measured with Fluorescence spectrophotometry trophotometer

on P-

previously with 0.1 N acetic acid. When the compound moved in one orange-red band and

eluted with water, the eluate gave one spot at 25 volts

by column chromatography

by the

simultaneous

addition

to the medium. On the other in cobalt-sufficient media.

absorption, CD and fluorescence ceus pigment. The absorption

of

100 g of the

cells

It was increased glycine, succinate

hand,

such a compound

Figure 1 shows the spectra of the puried S. olivaspectrum indicates its close simi-

larity

to that of Chromatium Co-free corrinoids reported by Toohey Although the wavelengths of absorption maxima for the 2S (1) (2). olivaceus compound coincide with those of the Chromatium compounds, the absorption ratio are different, especially in the ultraviolet region. It appears likely that this discrepancy may be due to a delicate difference between the structures of these compounds in minor details. By lacking the absorption around 400 mu, the eS olivaceus compound can be clearly distinguished from porphyrins. 171

Vol. 39, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-7 I, L w 4 d u'

1 d ci I.(

+20-

0.! \ 0

0. '\\

I' \ -to-

I 300

230

1

I 500

400

Wave

1

\-.--.&

1' \ : 1' ':

-2o-

Fig.

/ ,'

'1

' \\

length

1 600

( mp)

Absorption, emission and CD spectra the S. olivaceus compound.

of

The emission spectrum was measured relative to excitation at 500 mu. The concentrgsion of the compound was approximately 3.4 X 10 M.

I.0 -

S.ol ivoceus compound .:.:

I

2

3

4

5

6

8

7

9

IO

II

12

PH

Fig.

2

Paper electrophoretic compound at varied

pH.

behavior

of the

S. olivaceus

The experimental conditions are described in the text. The Chromatium compound cited as a reference is a Toohey's "positive" Co-free corrinoid (2). The CD and fluorescence with

those

of the Co-free

spectra

of

corrinoids 172

this

pigment

are also

of Chromatium

(5)

identical

and obviously

BIOCHEMICAL

Vol. 39, No. I,1970

different (7). that

from

those

These facts S. olivaceus

striking phoresis.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of porphyrins

(6)

and Co-containing

corrinoids

offer additional evidence for the possibility pigment is a kind of Co-free corrinoid. The

feature of this compound was the behavior in paper electroFigure 2 shows the results obtained by the method of

The S. olivaceus compound exhibits a more acidic Ladd et al. (8). behavior than the Chromatium compound. Judged from the degree of dissociation at neutral and alkaline pHs, it seems probable that the

carboxylic

amidated

yet

acid unlike

side-chains

of its

the Chromatium

corrin

nucleus

are not

compounds.

DISCUSSION The pigment

produced

by S. olivaceus

605 gave the

spectral

behaviors characteristic of the Co-free corrinoids of photosynthetic bacteria (1) (5). This compounds could not be detected when the organism was cultivated in cobalt-sufficient media. Addition of glycine,

succinate

and methionine,

the precursors

of corrin

enhanced the yield of the pigment in the cobalt-free nucleus, medium used. Paper electrophoretic studies performed at various pH indicated a more acidic character of this compound than the Chromatium

compounds.

From these

results

it

seems reasonable

to

conclude that the S. olivaceus compound is a Co-free having free carboxylic acid side-chains on its corrin

corrinoid nucleus

and,

hence,

biosynthesis

than than

the Chromatium the Chromatium

located

color turned into attempt to insert

at an earlier compounds. compounds.

stage

of corrinoid

This compound was more unstable In alkaline conditions its reddish

yellowish like the Chromatium cobalt atom into the yellowish

compound, but the compound performed

as mentioned by Toohey (1) was unsuccessful. The reason was considered that the conditions, such as boiling, employed for insertion

of cobalt

would not be appropriate

for

this

the

unstable

compound.

REFERENCES 1. 2. 3.

Toohey, J. I., Proc. Natl. Acad. Sci. U. S., 54, 934 (1965) Toohey, J. I., Fed. Proc., 25, 1628 (1966) Sato, K., Shimizu, S. and Fukui, S., Agr. Biol. Chem., ( in press ) 173

Vol. 39, No. 1, 1970

4.

5.

BIOCHEMICAL

Sato, K., Shimizu, 32, 1 (1968) Thomson, A. J., J. R. J.,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

S. and Fukui, Amer.

6. 7.

Porra,

Biochim.

8.

Ladd, J. N., Hogenkamp, Chem,, 236, 2114 (1961)

S.,

Chem. Sot.,

Biophys.

Acta

H. A. 0,, Pratt, Firth, R, A., Hill, P. and Jackson, W. R., Biochemistry,

Agr.

Biol.

2780 (1969)

91, 107, J, 6,

176 (1965)

M,, Williams, 2178 (1967)

H. P. C. and Barker,

174

Chem.,

I-L A.,

R. J. J.

Biol.