Vol. 168, No. 3, 1990 May

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

16,199O

TARGET

Hiroko

Department

Received

OF

Hama.

of Okayama

April

INHIBITION IN

SERINE

Yukiko and

Sumita, Tomofusa

Microbiology, University,

Yuri

Escherichia

Kakutagi, Tsuchiya

Faculty Tsushima,

of

1211-1216

coli

Masaaki

Pharmaceutical Okayama 700,

Tsuda

Sciences, Japan

7, 1990

SUMMARY: L-Serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, P-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serineresistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I. O1990 Academic Press,

Inc.

Although coli

cells,

For

example,

medium

amino some

synthase.

(2).

and

and

cause

similar

serine

has

it

inhibits

This

inhibition

is

the

not

taken

becomes

high

pathway(s). one

is

*To

whom

to

add

actively

(5).

Then ways

glycine

correspondence

is

released

amino

acids

by

by

by

the

serine

cells

known

and

leucine

should

to

release to

be

serine

the

the growth

added

known

for

the

medium.

35

years

inhibition

to by

culture

intracellular may

the

over of

to

culture

biosynthesis to

target

added

to

acetohydroxy

valine)

isoleucine

the

its

conditions.

of

(and

is

and

when

inhibition

adding

Escherichia

certain

K12

its

but

intracellular

are

coli

been

have

for

under

isoleucine

When

this

--E.

(3,4).

identified.

up

of

in

nutrients

growth

caused

inhibition

been

Two

growth

involved

inhibition

growth

their

is

other

excellent

inhibit the

is

some

usually

acids

which

so

L-Serine

amino

are

valine

(1).

acid

acids

medium,

.concentration

inhibit

growth

inhibition

medium

(6).

some

metabolic by

and

the

serine: other

is

addressed.

ooo6-291x/90 1211

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 168, No. 3, 1990

to

add

serine

isoleucine

(3.7).

deaminase

(6).

deaminase

rapidly

inhibition. that

biosynthetic the

target

and

of

perhaps

the

glycine

of

target

pathway,

like

that

serine

inhibition.

by by

for

MATERIALS

AND

so

of

is

by

valine.

of in

L-

L-serine

releasing

addition

serine

inhibition

induces

level

and

inhibition

inhibition

leucine

high

serine,

growth

for

and

resulting

intracellular

release

the

of

addition

metabolizes The

suggests

The

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

growth isoleucine

the

isoleucine

Here

we

report

METHODS

Bacterium and Growth Cells of E. coli W3133-2 (B), a derivative of K12, were grown aerobically in a mifima~edium (9) supplemented with 40 mM potassium lactate at 37°C. Serine-resistant mutants which grew normally in the presence of 1 mM L-serine were obtained from W3133-2 with nitrosoguanidine mutagenesis (10). Preparation of Homoserine Dehydrogenase I Homoserine dehydrogenase I was prepared from cells grown in minimal medium (9) supplemented with 40 --E. coli mM potassium lactate by a published procedure (11). The enzyme was partially purified by ammonium sulfate fractionation (precipitate at 37 to 50X saturation). Enzyme Assay Homoserine dehydrogenase activity was measured by reverse direction assay (13). The reaction mixture contained 0.1 M potassium 0.25 mM NADP, 10 mM MgNa -EDTA, 10 mM homoserine phosphate buffer (pH 7.2). and enzyme. The reduction of NADP was measured by a$I sorbance changes at 340 nm. A blank for NADP reduction was obtained by omitting homoserine.

When with

cells

lactate

of

--E.

a

sole

as

strongly

inhibited

intermediary

the

Krebs

etc.

(Fig.

released

and

cycle)

of

by

The

addition

of

but in

but addition

not

the

by

is

synthesized

by

and

that

not

isoleucine,

are

isoleucine

alone

of

1).

least

minimal

amounts

synthesized could

coli

cells

1212

by

acid,

is

results located of

even restore

(a

of on

growth

of

acid, serine

was

threonine suggest between

lysine, when

the

member

P-ketobutyric

E.

mM)

effects

oxaloacetate

These

serine

(1

pathway

2-ketobutyric (Table

the

threonine, of

supplemented

L-serine

tested

from

inhibition

inhibited at

We

of

biosynthetic

homoserine,

aspartate

medium

addition

1).

isoleucine

isoleucine,

minimal

an

the

growth

pathway

in

(Table

aspartate,

(2.8).

DISCUSSION

carbon,

growth of

via

1)

homoserine,

because

cultivated

Isoleucine

site

threonine,

were

source

metabolites

homoserine, main

AND

coli

the

serine-inhibition.

RESULTS

or that

aspartate

methionine serine (see

the

and

is

Fig.

added.

1).

Vol.

168,

No.

Table

3, 1990

1.

BIOCHEMICALANDBIOPHYSICALRESEARCH

Release

of

Serinea (1 mW

serine-inhibition oxaloacetate-isoleucine

by

Additiona

+ + + + + +

aspartic homoserine threonine P-ketobutyric isoleucine

COMMUNICATIONS

intermediary pathway

metabolites

Growth (doubling

acid (0.2 mM) (0.2 mM) ( 0.1 mM) acid (0.1 (0.05 mM)

mM)

of

the

rateb per hour) 0.74 0.00 0.00 0.58 0.61 0.66 0.61

aThe indicated concentrations of L-serine and intermediary fop) were added to the culture medium. Cells of E. coli W3133-2 were cultivated aerobically supplemented with 40 mM potassium lactate at 37°C. monitored turbidimetrically at 650 nm.

metabolites in

(L-

a minimal medium Cell growth was

oxaloacetate f aspartate f aspartyl f lyslne

e-------

e-----

klnrse

I, II, Ill

phosphate aspalate

aspartate c

methlonlne

asparlate

semlaldehydo

dehydrogenase

semlaldehyde homoserlne

dehydrogenase

I, II

homorerlne

Serlne . k

fl Titre&in~

100 80

h

thmonlne

2-ketobutyrlc f

deaminase

60

ackt

acetohydroxy

40

acid synthase 20

02 arkowheads

Metabolic Relevant indicate

pathway intermediates multistep

from

oxaloacetate and enzymes processes.

are

01. 0

‘.

”

2

4

’ 0.0

Concentration

to isoleucine shown. Broken

in E. lines

.

(mM)

coli with

Effects of serine and threonine on homoserine dehydrogenase I. Fig. 2. Fo;o)serine dehydrogenase I was prepared from E. coli W3133-2 (0). or SR33 , a serine-resistant mutant derived from W3133-2. The indicated concentrations of L-serine or L-threonine were added to the assay mixture. Control value (100%) for homoserine dehydrogenasf activity in-tlhe wild type and mutant was 12 nmoles NADPH formedemin.mg protein . Similar serine(and threonine)-resistance in homoserine dehydrogenase activity was observed in seven other mutants.

1213

’

0.1

’

.

’

0.2

Vol.

166,

No.

There first

are

step,

three

steps

conversion

of

aspartate

kinase

phosphate

to

and

another

homoserine

aspartate is

kinase

very

low

the

I

kinase

we

tested

I

homoserine

I

serine

and

lower

I

Thus,

the

by

target

I.

dehydrogenase

lactate

of

isolated even

serine

was

serine is

the

The

activity

wild

type

for

I

activity

II

activity of

E.

the

coli

K12

dehydrogenase

activity,

inhibits

aspartate

inhibits

homoserine

threonine (17).

cysteine aspartate

and

on

the

III

kinase

cysteine,

homoserine

serine

may

I

dehydrogenase activities

homoserine

No

of

(17). inhibit

activity.

aspartate

dehydrogenase showed

kinase

not seems

interesting

that whereas (Fig.

as

be

to

serine

reported

the

aspartate

mutants

that

(17).

homoserine

inhibited

and

at aspartate

inhibited

threonine 2)

of

shown),

by

inhibition

inhibition

(data

inhibition

I activity

stronger

significant

observed

activity

only

the

both

the

kinase

activity

enzyme.

eight in

(13).

responsible

kinase

in

homoserine

Threonine

activity,

the

II)

inhibits

serine

2). serine.

It

aspartate (15).

to

of

than

of

homoserine

We

activity

is

II

and

dehydrogenase

the

for

I

aspartyl

I.

(Fig.

activity

shown)

homoserine

lysine

inhibition

threonine

dehydrogenase

not

the

and/or of

strong

homoserine

(data

protein)

threonine

dehydrogenase

dehydrogenase

semialdehyde

I)

and

effects

concentrations

kinase

aspartate

pathway,

activity

the

We observed

of

(bifunctional

of

important.

resembles

kinase

Thus,

conversion

respect

(12).

structurally

aspartate

semialdehyde

dehydrogenase

dehydrogenase

activity

conversion

by

aspartate

dehydrogenase

is

catalyzed

by

II

I

step,

is

The

catalyzed

II

with

phosphate,

1).

is

dehydrogenase

Thus,

(Fig.

second

protein

(homoserine

homoserine

homoserine

and

COMMUNICATIONS

the

step,

activity

and

aspartyl

bifunctional

(homoserine

serine

and

third

aspartate-homoserine

dehydrogenase As

(13);

by

dehydrogenase

In

to

protein I

(16).

homoserine

III

the

single

kinase

and

(14).

aspartate

aspartate

catalyzed

a

aspartate

between

and

and is

Interestingly,

BIOPHYSICALRESEARCH

semialdehyde,

(13);

homoserine,

the

II

I,

aspartate

dehydrogenase to

BIOCHEMICALAND

3, 1990

presence

serine-resistant of

1 mM serine 1214

(data

not

shown),

grew

normally and

tested

on the

Vol.

BIOCHEMICAL

168, No. 3, 1990

effect

of

whether

serine homoserine

serine.

As

mutants that

on

was serine

their

homoserine

expected,

the

resistant

to

I

is

homoserine serine

growth

RESEARCH COMMUNICATIONS

I

dehydrogenase

dehydrogenase

causes

AND BlOPHYSlCAL

the

real

target

dehydrogenase

and

by

(Fig.

determine

inhibition

activity 2).

inhibiting

to

for

I

threonine

inhibition

activity

in Thus,

by

all we

homoserine

these

conclude

dehydrogenase

I. There

is

a

report,

I

dehydrogenase

in

however, --E.

coli

that (14).

serine We

does

do

not

not

know

inhibit the

homoserine

reason

for

this

discrepancy. As not

the

inhibition

complete,

activity

and is

low

(16).

amounts

is

isoleucine

is

also

a

involved

interfere

with

expression

of

biosynthesis reasons

from for

homoserine

of

and

the

threonine-isoleucine deamination

for

the

growth

restored

cell

which

and

However,

these

inhibition,

because

branched

addition

Thus, at least Then,

conditions? is

the

may chain

indirectly effects

its

aspartate

synthesized.

these

and

and

so

also

repress

amino

serine of

why Serine

first

with of

is

although

1).

Serine

of

directly

threonine.

its

(7.18).

serine

from

pathway,

syntheses

interfere

be

under

deaminase,

by

present,

Fig.

would

threonine

activity

synthesized

(see

threonine

from

is

be

serine

threonine

operons may

would

for

threonine

serine

presence

synthesized

in

II

homoserine

methionine

substrate

enzyme

Thus,

the

I

dehydrogenase

dehydrogenase

some

in

of not

homoserine

homoserine

even

semialdehyde minimal

of

it

acids

key will the (7).

isoleucine are

not

main

threonine

growth.

REFERENCES

:: 3. 4. 5. 6. 2 9.

Tatum, E. L. (1946) Cold Spring Harbor Symp. Quant. Biol. 11. 278-284. Umbarger, H. E. (1987) in Escherichia coli and Salmonella typhimurium (F. C. Neidhardt ed.), Vol. 1, pp. 352-367. ASM, Washington,DC. Amos, H. and Cohen, 6. N. (1954) Biochem. J. 57, 338-343. Alfoldi, L. and Kerekes, E. (1964) Biochim. Biophys. Acta 91, 155-157. Hama, H., Shimamoto, T., Tsuda, M. and Tsuchiya, T. (1987) Biochim. Biophys. Acta 905, 231-239. Newman, E. B. and Walker, C. (1982) J. Bacterial. 151, 777-782. Uzan. M. and Danchin. A. (1978) Mol. Gen. Genet. 165, 21-30. T. and Wilson, T. H. (1978) J. Bacterial. 134. Lopilato. J., Tsuchiya, 147-156. Tanaka, S., Lerner, S. A., and Lin, E. C. C. (1967) J. Bacterial. 93, 642-648. 1215

or

Vol. 168, No. 3, 1990

10. 11. 12. 13. 14. 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

M. and Chen, G. C. C. (1965) Biochem. Biophys. Adelberg, E. A., Mandel, Res. Commun. 18, 788-795. Truffa-Bachi. P. and Cohen, G. N. (1970) Methods in Enzymol. 17, 695699. Datta, P. (1967) Proc. Natl. Acad. Sci. USA. 58, 635-641. Cohen, G. N. and Saint-Girous, I. (1987) in Escherichia coli and Salmonella typhimurium (F. C. Neidhardt ed.) Vat. 1, pp. 429m. ASM, Washington,DC. Patte, J. C., Truffa-Bachi, P. and Cohen, G. N. (1966) Biochim. Biophys. Acta 128, 426-439. Patte, J. C., LeBras, G. and Cohen, G. N. (1967) Biochim. Biophys. Acta 136. 245-257. Falcoz-Kelly, F. and Cohen, G. N. (1970) Methods in Enzymol. 17. 699702. Stadtman, E. R., Cohen, G. N., LeBras, G. and Robichon-Szulmajster, H. (1961) J. Biol. Chem. 236, 2033-2038. Rasko, I. and Alfoldi, L. (1971) Eur. J. Biochem. 21, 424-427.

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