The biosynthesis of magnamycin, a macrolide antibiotic

BIOCHEMICAL

Vol. 8, No. 4, 1962

AND BIOPHYSICAL

THE BIOSYNTHESIS A MACROLIDE Dorothy

Gilner

OF MAGNAMYCIN, ANTIBIOTIC*

and P. R. Srinivasan

College of Physicians and Surgeons, of Biochemistry, Columbia University, New York, N. Y.

Department

Received

Junel2,

1962

The successful the

RESEARCH COMMUNICATIONS

macrolide

antibiotics

the biosynthesis

of

member

of this

lactone

ring

to which

The lactone

branching substituents,

as well taken

has been

suggested

be formed ation

into

the

starch,

in yeast

were

then

the

above

present

architecture

by a large

disaccharide

mycaminosylmycabackbone

group,

with

three

and an epoxide

unique

in

is an inter-

characterized

aldehyde

of

interest

Magnamycin

a 17 carbon

as a carbonyl

lactone

extract,

composition,

of

a

hydroxyl

ring

(1).

These

biochemical

problems

and it

the

macrolide

antibiotics

may

and propionate

(1).

that

incorporation

a medium

separated

the

a branching

acetate

Streptomvces 60 hours

attached

chemical

considerable

of compounds

by Noodward

the

the

compounds.

consists

together

from both

we report

residues

is

group,

features

novel

class

ring

methyl

of

has stimulated

these

esting

rose.

elucidation

of both

moiety

propionate

was grown of

casamino

the

acids,

by centrifugation, and again

this

communic-

and acetate

of magnamycin.

Halstedii consisting

In

placed

on a rotatory

following and CaCO9.

The mycelia

resuspended

in

on the

for

components:

shaker.

*This investigation was supported by a grant from Institutes of Health, United States Public Health RG-5809. 299

shaker

a medium

of

Under

these

the National Service, No.

Vol. 8, No. 4, 1962

conditions

BIOCHEMICAL

the

8 to 10 hours imately

accumulation after

tation lated

eral

after

essentially

the

compounds

20 hours

worked (3,4,5)

stant

point

pounds was also

and sufficient

out

during

(Fig.

1).

established

counts

are expressed weight

of

"C-12

(2).

of

Fig I : Scheme

per minute

the degradation

300

structure

of

Magnamycin

to conof the

steel

error

multiplied

and are summarized

gen-

com-

spectra.

on stainless

MYCAROSE

for

the

the

The purity

MAGNAMYCIN

ISOVALERYL

fermen-

The degradation

followed

a probable

were

formed was iso-

and ultraviolet

to give

as counts

and the

acid"

activity.

thickness

the canpound

al.

liter)

compounds were purified

by infrared

were taken

et

the elucidation All

at infinite

(24 mmoles per

The magnamycin

and to the

at

a maximum at approx-

had started

later.

and to constant

Samples were counted

molecular

and reaches

accumulation

to carimbose

of magnamycin

The results

in the medium begins

by the method of Tanner

procedures

melting

of magnamycin

The labelled

was terminated

of magnamycin

RESEARCH COMMUNICATIONS

resuspension

40 hours.

added two hours

AND BIOPHYSICAL

in the

of

dishes 5 percent.

by the Table.

BIOCHEMICAL

Vol. 8, No. 4, 1962

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Incorporation of Acetate-2-C14, Propionate-2-C14 and Propionate-l-C14 into Magnamycin and its Degradation Products Activity

Compound

in

the various experiments (c.p.m. x M.W.)

Acetate-

Propionate-

Propionate-

2-cl4

l-Cl4

2-cl4

Precursor

357

x 104

316

x 104

474

x 104

Magnamycin

293

x 104

156

x lo4

265

x lo4

Carimbose

265

x lo4

151 x 104

258

x lo4

"C-12

105 x 104

--

255

x lo4

acid"

Fatty acids* (Lithium salts) * Figures

salts in

in

the

represent

counts

The fatty

acids

the

Table.

ciently

4060

various

incorporated

existence ganism.

of

fatty

but

is

it

is

fatty acids

also activities

clear

that

acids. adduces

significant the

are

presented

acetate

evidence

is

to

the

pathway

in

activity

occurrence

as lithium

effi-

The incorporation

CoA * succinate

yet

isolated

of

from some

of probable this

or-

propionate-l-

fatty

acids

which

propionate.

approximately

pionate-2-C14. pionate-l-Cl4

were

and their

the

The incorporation mycin

cells

results

seem to suggest

incorporate

the

the methylmalonyl

The small

Cl4 would

of

into into

258

per minute.

experiments

From the

propionate-2-Cl4

878

of twice

The similarity and -2-C14

into

label

that in

from from

the

magnamycin

acetate-2-Cl4

propionate-l-Cl4 percent

incorporation

and carimbose

into

magna-

and proof prosuggests

that

Vol. 8, No. 4, 1962

propionate

BIOCHEMICAL

is

acid"

obtained

l-Cl4

has

introduced

as an intact

on degradation

the

same molar

indicates

that

propionate

tent

into

the

must

contain

sugars

Grisebach

tained

from

mycin

isolated as the

the

observations that

of Grisebach "C-12

the

the branching

methyl

group

of

the

contains imply

that

"C-12

experiment

acid"

are in and it

acid"

acid from

ob-

magna-

same activ-

agreement

is

with

reasonable

one propionate being

ex-

was in pro-

had the

experiments

only

"C-12

acetic

to

residue,

derived

from

the

with

The

ring.

of

the

activity

(i.e.

12 to 18.

left

lactone

remainder

lost

incorporated

reside

during

the

degradation.

the

"C-12

The lactone

methyl one assumes the

must

into

one aldehyde

that

the branching

ring

16 carbon

atoms

acid"

are derived of

the 302

lactone

"C-12 This of

the

that

as into

of 17 carbon

ring

atoms,

and an 0-acetyl

methyl from

would

indicates

as well

group

acid"

12 to 18 and in

This

consists

group,

lactone

atoms

from of the

60 percent

in carbon

ring

acid“

on the biosynthesis

of carimbose.

carbons

10 and 9 of

the

"C-12

activity

group

If

of

light

to the

the

is

group.

carimbose

sheds

skeleton

of carimbose)

one branching

the

of

acetate

is

the

the

40 percent

the

acetyl

of

experiment

carbon

only

activity the

work

contains

This

of propionate.

acetate-Z-Cl4

rest

this

of magnamycin

The degradation the

While

and Achenbach

acid"

3).

to any significant

(6) showed that

Thus our

propionate-

(Column

the

a CT3 -C14H2-C00H

acid".

magnamycin

that

of

The "C-12

from

and also

residues.

oxidation

unit.

derived

incorporated

and Achenbach

"C-12

three-carbon

as the

is not

the Kuhn Roth

ity

methyl

activity

or isovalerate

from

RESEARCH COMMUNICATIONS

of magnamycin

the propionate

gress,

suggest

AND BIOPHYSICAL

group

and carbons

propionate, ring

(including

then the

one

Vol.

8, No.

4, 1962

aldehyde arise

group) from

cent

BIOCHEMICAL

two carbon

5/9

x 100)

acetate-2-Cl4

It

should

The actual

is

atoms

therefore of

of

and Achenbach

mode of

formation

ing

hypothesis

(Fig.

functions

as well

magnamycin.

of the 2).

as the

in biological

sation

between

in

This

of

* Since mycarose mycaminose ** Our suggests malonyl-CoA thesis of

lactone

acetate and propionate moiety of magnamycin, is also not derived

rest

into

double

the

interesting

Scheme ring

account bond

: acetate

R:

prapianate

for

the

of

Magnamycin

the

follow-

the

oxygen

observed

carbon-carbon questions.

as that

as a work-

to an aldehyde

P

carbon

as well

of magnamycin

the

from

60 percent.

the

to advance

takes

56 per-

"C-12

is of

atoms

to acetate?*

like

However,

CH,-CH,-C

of

derived

findings

ring

C-C

the

the

of a carboxyl

Hypothetical

a loss

to the

origin

scheme

position

18 carbon

our experiments

that

lactone

reactions.

of

then

experimental

C-6 and C-7 raises

Fig 2:

these

degradation

(6) we would

The reduction

common

its

may owe their

of our

COMMUNICATIONS

of carimbose*

to suggest

On the basis

ing

activity

obtained

ring

If

of acetate,

during

figure

lactone

of Grisebach

the

RESEARCH

group.

units

occur

tempting

the

BIOPHYSICAL

one 0-acetyl

the

(i.e.

acid".

plus

AND

in

is not

un-

condenAn altern-

unit unit

biosynthesis

are poorly incorporated into the it is reasonable to assume that from these precursors.

current understanding of the biosynthesis of fatty acids that acetate and propionate will probably participate as and methylmalonyl-CoA, respectively, in the biosynmagnamycin. 303

Vol. 8, No. 4, 1962

ative

possibility

a four ruled are

BIOCHEMICAL

carbon out

that moiety

by our

in progress

carbon which

to answer

Pfizer

and also

for

& Co., the

Inc.,

generous

atoms

in turn

experiments

We are extremely Chas.

AND BIOPHYSICAL

is

at the these

and 8 may be derived

formed

present

intriguing

grateful for

5,6,7

from

acetate

time.

from

cannot

be

Investigations

problems.

to Dr.

a culture

supply

RESEARCH COMMUNICATIONS

F. A. Hochstein

of Streptomyces

of

the

Halstedii

of magnamycin.

References 1. 2. 3. 4. 5. 6.

woodward, R. B., in Festschrift Arthur Stoll, Birkhtiuser, A.G., Basel, 1957, p. 524. Tanner, Jr., F. W., Lees, T. M., Routien, J. B., U.S.Patent, 2,796,379 (1957). Wagner, R. L., Hochstein, F. A., Murai, K., Messina, N., and Sot. , 75, 4684 (1953). Regna, P., J. Am. them. Breslow, R. c. D., Thesis, Harvard University (1955). Harvard University (1957). Agosta, W. C., Thesis, Crisebach, V. H., and Achenbach, H., 2. Naturforsch., 17, 63 (1962).

304