Altered folate metabolism in a vitamin B12-methionine auxotroph

Vol. 16, No. 6, 1964

BIOCHEMICAL

ALTERED FOLATE METABOLISM Herbert

convergence

zymatic

moieties.

steps

and Woods

and cofactor

(1953;

of methionine

1960),

found

carbon

precursors

methionine

The object

two individual

methionine-methyl

groups for

methionine-vitamin

of methionine. growth

the group

homocysteine of the enWijesundera

recognized

that

led

to the

Rowbury

both

a

synthesis,

conversion

occurred

study enzymes

the

suppression and Woods

of various

--in vivo

one-

and in vitro.

in methionine-free

was to determine involved

During

exists.

of the growth

rate

auxotroph

cyano-B 12 for

growth

The following

results

what

in the

from N 5 ) 10 -methylene-folate-H

B 12 (cyano-B12)

sufficient

(1953),

is

media

of the organisms.

autoregulation

inhibition

coli

elucidation

and serine.

methyl

synthesis

present

of the

k-methionine

repress

of growth

of the

had on the

a paradoxical

containing

to the methionine

to resumption

locus

as Cohn --et al

could

group

those

of methionine

from homocysteine

of methyl

even prior

observed

Heart Institute Maryland

of Escherichia

namely,

requirements

in media

that

Derepression

tions

National Bethesda,

to the complete

as well

synthesis

(1961)

containing

ADXOTROPH

Weissbach

in strains

syntheses;

Even prior

of organisms

specific

of methionine

of two separate

and methyl

onine

B12 -METHIONINE

and Herbert

Biochemistry, of Health,

RESEARCH COMMUNICATIONS

June 24, 1964 The biosynthesis

growth

IN A VITAMIN

Dickerman'

Laboratory of Clinical National Institutes Received

AND BIOPHYSICAL

4 the

formation

but are

with

methiof the

and whether

course

was observed of E . coli

effect

of this

during

593

of a

K12 in a medium

suboptimal

an attempt

of an Established

study,

growth

concentra-

to clarify

inhibition.

1 This study was done during the tenure of American Heart Association, Inc.

a

Investigatorship

this

Vol. 16, No. 6, 1964

BIOCHEMICAL

The organism E.

coli

strain

K12,

thiamine-)

which

National

used

in this

was generously

were

salts-glucose

medium

supplemented

and cyano-B12,

0.01

tion

56 described

on a rotary

transferred

10 minutes

fragmented clear

in

slants

1964).

into

C. twice

preparawere

incu-

Appropriate

aliquots

with

and

media,

100 ml minimal

concentrations

minimal

0.2 pg/ml,

The cultures

at 35'

media,

1 ml

of L-methionine =

by measurement

collected

once with

of turbidity

same buffer, with

probe

for

HCl buffer,

and disrupted

a Brownwill

by exposure sonifier.

at 5000 x g x 10 min.

used as source

concentration

by centrifugation

10 ml of 0.1 M Tris

was centrifuged

fraction

The protein

assays.

agar

(1951).

washed

were

of sonication

suspension

supernatant

al

was followed

2 ml of the

periods cell

(Taylor,

was a half-strength

contained

the cells

at 5000 x g, washed

two 15-second

the

of

DU spectrophotometer.

assays,

suspended

Taylor

media

harvested,

growth

broth

hours

which

12 '

hydrochloride,

and varying

Bacterial

For enzyme

pH 7-4,

were

I,.

or cyano-B

thiamine

et --

sixteen

flasks

at 600 q~ on Beckman

with

Austin

mutant,

and Blindness

tryptone

by Monod

1 pg cyano-El2

pg/ml).

by Dr.

The minimal

for

culture

to 1 liter

20% glucose, (O-1-20

pg/ml.

shaker

of the overnight

from

produced

2276 (methionine-

Diseases

transferred

RESEARCH COMMUNICATIONS

was the phage

provided

of Neurological

The organisms

bated

study

AB 1172 - substrain

Institute

of medium

AND BIOPHYSICAL

of enzyme(s)

of the

in the

supernatant

to The

and the

subsequent

fraction

ranged

from

1 - 10 mgm/ml. The assay been

described

of N5 -methylfolate-H in a previous

methylene-folate-H dependent hyde

oxidation

(Donaldson

equillibrium assay

4

is based

publication

reductase

the

on the

H4 on a Dowex 1 chloride

was assayed

1961).

immediate

product,

retention

of the

column

with

594

methyltransferase

(Weissbach,

of N5 - 14 C-methyl-folate-H and Keresztesy,

with

4 -homocysteine

by the back

et al.,

1963).

reaction,

to folate-H4 4 The latter compound

has

the menadioneand 14C-fonualdeis

in direct

N5~10-methylene-folate-H4. substrate

elution

N5,1%

The

14C-methyl-N5-methyl-folate-

of the ultimate

product,

14C-

BIOCHEMICAL

Vol. 16, No. 6, 1964

formaldehyde. wmole

A unit

of product

centration

formed

in

was determined In order responsible

inocula

of cyano-B12

per

grown

cyano-B12

synthesis

activity

mgm protein of Lowry

the effect

the

RESEARCH COMMUNICATIONS

specific

by the method

for

containing

enzyme's

60 min.

to demonstrate

enzymes

media

of either

AND BIOPHYSICAL

at 37' --et al

of exogenous

were

requirement

and graded

concentrations

effect

of methionine

on growth

was observed

Protein

con-

on the

moiety,

in glucose-salt

of the (Fig.

C.

methyl

twenty-fold

minimal

as 1

L-methionine =

subcultured

at a concentration

defined

(1951).

of the methionine

organisms

is

greater

amino

1).

minimal than

acid.

the

A marked

The cells

grown

on

--B,~fO5ppMclh/ml -B,Zt50,uqMelh/ml I I 2

D2 1 0

I 4

I 6

I 8

I IO

I 12

14

HOURS

Fig.

1.

cyano-B12 5 pg/ml cyano-B12 significantly cell

The inhibition of growth of E. coli K mutant 2276. The inocula were grown in a glucose-salt minimal m cyano-B12 l$ dia containing The subsequent subcultures were grown in the same (0.01 &ml). media with the following additions: -, cwo-B83il;.0~;, ocyano-B12, 0.01 ug/ml plus L-methionine 0.5 p 0.01 pgfml plus &-methiozine, 5.0 yg/ml, Growth was as the change in turbidity recorded at 600 mp; temperature was 35O C. alone grew

or in combination

at the

same rate

and an exogenous

divisions.

inhibited, Further

with

and to the

methionine This studies

methionine

same extent.

concentration

inhibition revealed

595

at concentrations Those

exceeding

cells

grown

of O-5 - 2.5 pg/ml

became apparent

after

that

inhibition

the growth

with were

one to two was

Vol. 16, No. 6, 1964

dependent added

during

added

after

BIOCHEMICAL

on the

time

of addition

the

first

two hours

this

when glucose

was the

Extracts for

N5j1'

interval, carbon

of growth,

source grown

-methylene-folate-H

4

methyltransferase

activities.

exceeded

4 kg/ml,

there

activity2

(Fig.

but

under reductase

was almost in

METHIONINE

not the

the amino

with

would

The growth

was

result, effect

were

and N5-methyl-folate-H of exogenous

4

methionine

suppression

the concentration

acid

but

if

was observed

glycerol.

above conditions,

complete

/q/ml

If

inhibition

was seen.

At levels

However,

RESEARCH COMMUNICATIONS

of methionine.

no effect

of cells,

2).

AND BIOPHYSICAL

range

assayed -homocysteine which

of the reductase of 0.5

- 2.5

MEDIA

5,lO reduc-methylene-folate-H Changes in specific activity of N Conditions of tase with varied media methionine concentrations. growth and preparation of the extracts are described in the text. The reaction mixtures contained KBO buffer, pH 7.4, 10 umoles; N5-14C-labelled methyl-folate-H4, 28' wmoles (specific activity 2000 c.p.m./ngrmole); flavin adenine dinucleotide, 1 mumole; menadione bisulfite, 1 *mole; sodium ascorbate, 5 umoles; formaldehyde, 5 umoles; and suitable aliquots of enzyme. Final incubation volume was 0.2 ml. Duration of incubation was 30 min. and temperature was 37O C. The control tubes contained all the reactants except enzyme; the enzyme was then added at the end of the incubation. Following incubation 0.3 ml of ice water was added to all tubes and the contents of the tubes were applied to a Dowexl-chloride column (0.5 x 3.0 cm). The eluate as well as a 1.5 ml water wash was collected in a vial for liquid scintillation counting (Brav. 1960). 5,lO -methylene2 Katzen has reported that a marked derepression of the N folate-H4 reductase occurred in E. coli W 113-3 when the cells were grown on cyano-B12 alone or with low concentrations of L-methionine (Katzen, = 1964). Fig.

2.

596

BIOCHEMICAL

Vol. 16, No. 6, 1964

pg/ml

of methionine,

activity There

compared

there

was a five-to-ten

to extracts

was no evidence

AND BIOPHYSICAL

derived

of --in vitro

RESEARCH COMMUNICATIONS

fold

increase

from cells

activation

grown

of the

in reductase

on cyano-B

reductase

alone.

12 activity

by

20

06 .05 04

01

Fig.

3.

addition

of methionine. was observed

in reductase

activity

to the alterations led

a comparable

activity

I

I

3

4

I

I

I

5 67 HCURS

I

I

8

9

.,

IO

Protective effect of nucleosides on growth inhibition observed at "low" methionine concentrations. The culture conditions were the same as described in Fig. 1 with the following additions: -, cyano-B12, 0.01 ug/ml: o- - -0, cyano-B 0.01 &ml, A?61 pg/ml, Lplus &-methionine, 1 pg/ml; #, cyano-B12, methionine, 1 ug/ml, guanosine, 0.1 pmole/ml and uridine, 0.7 umolefml. Growth was again determined as the change in turbidity recorded at 600 q.

inhibition

medium

1

I2

The concentrations were

in the

was observed in reductase

to an incomplete

same range (between

activity,

in specific

The association

of growth

of ii5,10 -methylene-folate-H

0.3

as those

at which at which

- 2,5 pg/ml).

the addition

suppression

increase

of methionine

growth increase

In contrast

of methionine

of methyltransferase

activity

to the without

activity. inhibition 4 597

with

reductase

an increased suggested

that

specific a divergence

Vol. 16, No. 6, 1964

BIOCHEMICAL

of one-carbon

units,

generated

might

be occurring.

group

synthesis

at the

a test

of this

hypothesis,

expense

added

methionine,

Fig.

3 shows

of guanosine inhibition.

centrations to the exogenous

second

nucleosides

Guanosine, adenosine, Deoxyguanosine

pyrimidine either

after

inhibition

had occurred

or in combination

effective

growth

inhibition.

alone

shunt

of one-carbon

units

purine

deficiency.

enzyme under

levels

of methionine

higher

levels

control late

these

as this and methionine

It

in the medium and almost

of the amino

the metabolism

conditions.

acid

are

present.

provides

one-carbon

biosyntheses.

598

adenosine,

alone

protective.

pyrimidine

nucleo-

situation

of the

in which

results appears

to be the

stimulated

completely

repressed

is

tempting

is under units

for

a

in a relative

is markedly

of N5,lO -methylene-folate-H4

compound

or

inhibition.

slightly

reductase

It

prior of

to an intensification

synthesis 4

added

by addition

other

an experimental

N%1O -methylene-folate-H

were

while

with

methionine

the

by low con-

growth

was only

led

against

nucleosides

the

as guanosine,

or actually

towards

Addi-

were unsuccessful.

against

nucleosides,

describes

experiment.

inhibition

pyrimidine

or in combination

ineffective

report

that

as effective

pyrimidine

The present

sensitive

in protection

or in of

at reversal

with

As

concentration

the nucleosides

Attempts

with

were

if

alone

the cells

the

division.

nucleosides

sides,

only

like

cell

was almost

or in combination

of a representative

methyl

synthesis.

nucleosides,

protected

reaction

methionine

an inhibitory

effect,

occurred

alone

was most

increase

base and thymidylate

to the media

The protective

or

would

containing

the results

of methionine,

first

shunt

hydroxymethylase

and pyrimidine

to media

and uridine

RESEARCH COMMUNICATIONS

the serine

of purine

purine

were

growth

through

Such an abnormal

combination,

tion

AND BIOPHYSICAL

by low when

to speculate stringent

purine

base,

metabolic thymidy-

Vol. 16, No. 6, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

REFERENCES Bray, G., Anal. Biochem., 1, 279 (1960). Cohn, M., Cohen, G. N., and Monod, J., C. R. Acad. Sci., Paris, & 746 (1953). Donaldson, K. O., and Kereszteey, J. C., Biochem. Biophys. Res. Commun., 2 289 (1961). Katzen, H. M., Fed, Proc, 23, 480 (1964). N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem., Lowry, 0. H., Rosebrough, 193. 265 (1951). Monod, J., Cohen-Bazire, G., and Cohn, M., Biochim. Biophys. Acta, 1, 585 (1951) . Rowbury, R. J., and Woods, D. D., J. Gen, Microbial., & 129 (1961). Acad. Sci., 50, 1043 (1964). Taylor, A. L., Proc. Natl. Weissbach, H., Peterkofsky, A., Redfield, B. G., and Dickerman, H., J. Biol. Chem., 238, 3315 (1963). Wijeeundera, S., and Woods, D. D., Biochem. J., 55, viii (1953). Wijesundera, S., and Woods, D. D., J. Gen. Microbial., 22. 229 (1960).

599