Vol. 88, No. 3, 1979

EIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 13, 7979

Pages Xl-767

PROPERTIES OF GLUTAMATE DEHYDROGENASE FROM BACILLUS SUBTILIS James F. Kane and Kathryn L. Deshpande Department of Microbiology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 Received

April

3, 1979

SUMMARY Bacillus subtilis strain 168 possesses an NAD-dependent glutamate dehydrogenase. The level of this enzyme is influenced by the the source of nitrogen, stage of growth, and a high rate of tryptophan biosynthesis. The enzyme appears to serve an anabolic function and, therefore, must be considered as a possible route for the incorporation of inorganic nitrogen into an organic form. INTHODUCTION In Bacillus

subtilis,

duced by the

concerted

and glutamate

synthase

1.

glutamine

action

3.

glutamine

+ a-ketoglutarate

enzyme involved

168 of g.

subtilis

that

enzymatic

of Bacillus.

of

sporulation

activity

As a result

the

+ NADPH ----->

2 glutamate

formation,

studies,

enzymes of (14,

+ NADH ---->

reported (l-3),

in several of

+ ADP + Pi

namely

+ NADP

glutamate

dehydrogenase

has been previously

activity

(E.C.6.3.1.2)

1.4.1.2.),

NH3 + a-ketoglutarate

ignored

synthetase

glutamine

in glutamate

(GDH; E.C.

glutamate

this

to be pro-

as follows:

+ ATP ------>

synthase

dehydrogenase

of glutamine

(E.C.2.6.1.53)

glutamate

Another

has been proposed

synthetase

NH3 + glutamate 2.

glutamate

glutamate

to be missing

although

there

exists the role particularly

+ NAD

in the competent have been reports

in other of this

strains

strain (2-5)

and species

enzyme has been

those

nitrogen

metabolism

761

Copyright All rights

that during

deal

with

the

the process

15). 0006-291X/79/110761-07$01.00/0 0 I979 bv Acudemic of reproduction in my

Press.

form

In (13),

anthranilate

has a defect of

+

and examined

for

in the trpE

the anthranilate

contains

the subunit

activity

excreted

into

contains

763

to 1

produced.

synthase

by the anthranilate

mutant,

tryptophan

selected

subunit

mutant

converted

in the anthranilate

designated

no tryptophan

utilized

The other

This

is

+ glutamine

the large

(13).

but

mole of

were therefore

essentially

no glutamine complex.

(EG)

complex

every

of NPlOO defective

One mutant,

the complex

a result

for

mutants

synthase

locus

one mole of glutamine

synthase a defective

G

--in vitro.

As

the medium, and enzyme subunit

G and

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Activity

of GDHin mtr s and mtr gat mutants derived from mtr mutant NPlOO

Collection No.

Relevant Genotypes

Specific Activitiesa Anthranilate synthase GDH E

G

-

0.05

NPl

5.0

NPlOO

mtr2

112

mtr2,

trpE5

115

e,

gat7

0.3

0.5

10.8

4.5

6.0

0.5

0

0

4.0

0.4

aThe specific activities of anthranilate/min/mg protein. uses ammonia instead tion

(7,

a factor

mutants

medium of mutants

level

rate

of glutamine

and is

tophan,

not

the anthranilate

the level

synthase

of GDH was reduced

(50 rig/ml)

These results

in GDH in the mtr mutant

a high

in

is

induced

is

suggest

a physiological

utilization

the

the

consequence

in the production

by the presence

in the

NPl,

that

reac-

by about

supplemented

112, and 115 or the prototroph

of GDH was unchanged.

increase of

glutamine

When tryptophan

of 10.

growth

of

In both

13).

are as follows: subunit E and G - nmoles protein; GDH- pmoles NADHoxidized/hr/mg

of tryptophan

of

tryp-

in the

medium. Helationship GDH.

In the prototroph,

reduced either

when the with

acid

one.

with

Glutamine,

supplement, In

cells

or without

more consistent lic

between

in repressing

NPl,

source the

of nitrogen

specific

and the level

activity

(NHq)2S04

(Table

a biosynthetic either

function

as a source or no effect

NPlOO,

however,

GDH than was glutamate

764

2).

of

glutamate

result

for

GDH than

nitrogen

glutamine (Table

of

This

on the

2).

of

of GDH is

are grown in the presence

had little

the mtr mutant

the

level

is a catabo-

or as an amino of GDH.

was more effective Presumably,

this

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE II Effect Nitrogen sourcea

of the nitrogen

source on GDH

Growth SuppIementb I__-

Specific Activity of GDH NPl NPlOO

(NH4)zSO4

None

6.60

5.2

(NH4)2S04

Glutamate

0.24

4.2

(NH4)$=4

Glutamine

0.45

2.6

Glutamate

None

0.24

2.6

Glutamine

None

0.60

1.1

aThe concentrations of the nitrogen sources were as follows: (NH4)2S04-15mM; glutamate - 0.2%; glutamine - 0.2%. bG1utamat.e and glutamine 400 ug:/ml.

is

related

to the

of

tryptophan

in

high this

were added to a final

utilization

of

concentration

glutamine

in

of

the production

mutant.

DISCUSSION These results

demonstrate

strain

168 possess

ties.

First,

stage

with

tionary

for

for

glutamate

the effect

vegetative Second,

species

growth

enters

fulfilling

sta-

been (2,5).

the

stationary

the cell's This lack

possibility

GDH and deter-

on ammonia assimilation

during

and sporulation.

GDH appears

on glutamate

in the early

of Bacillus

which

proper-

the growth

has also

sporulation. mutants

lesion

of

a maximum level

as the culture

by isolating of this

has the following

characteristic

during

of -B. subtilis

is a function

may be a mechanism for

be verified

mining

growth

reaching

in GDH activity

growth

requirement

activity

this

derivatives which

the GDH's from other

increase

phase of

could

activity

phase of growth;

reported This

a GDH activity

the specific the

that

to have a biosynthetic

represses

the activity

765

of

function this

enzyme.

since In

Vol. 88, No. 3, 1979

this

regard

tionship of

the

role

of this

Bacillus

the

is

indicated

by the

mutant

excretes

pool

for

glutamine

satory

effect

GDH activity effect.

That

than

in

the

112)

or gat

utilization These

results

to nitrogen

NPl).

in

(mutant

metabolism

tryptophan 0.9 the

biosynthesis

conversion

to alter

the mtr

mutant

which

the

a high

that rate

metabolites is

being

cause the of

and the

tryp-

levels

The altered

higher

rate

(mutant

of

a drop

increase

tryptophan

glutamine in GDH.

in GDH is a biosynthe-

relationship

of GDH

pursued.

ACKNOWLEDGEMENTS We thank Lynn Waltemath for her excellent assistance. This research was supported with Science Foundation grant PCM7718960.

technical funds from National

REFERENCES 1.

Freese, Acad.

E., Park, S. W. and Cashel, Sci. U.S.A. -51: 1164-1172.

766

of

a compen-

the trpE the

also

of

compensatory

decrease

hypothesis of

pool

perhaps

at either

excretion,

the

of

GDH is 5 to lo-fold

and mutations loci

of one mole

the

such a possible

dry of one

rate

metabolism.

suggests

NPlOO.

ug tryptophan/mg

a high

of nitrogen

this

(50 ng tryp-

enzymes

consequence in

by the

mutant

with

115)

support

the mtr

Since

to changes

metabolite(s);

and c-ketoglutarate

prototroph,

The changes

NPlOO verses

be expected

and tryptophan

physiological sis.

is,

of

might

of NPlOO

in species

to be responding

quantities

a-ketoglutarate,

on the

rela-

14).

of

glutamate

and the

of sporulation

of GDH in

is accompanied

biosynthesis

glutamine,

level

for

to one mole

tophan

(13,

prototroph

tryptophan

process

of some critical

large

weight/hr of

the

RESEARCH COMMUNICATIONS

metabolism

of GDH appears

high

dry weight/hr the

to

level(s)

tophan/mg

mole

nitrogen

level

in

This

of GDH in

be ignored

the

internal

AND BIOPHYSICAL

enzyme

cannot

Finally,

BIOCHEMICAL

M. (1964)

Proc.

Natl.

Vol. 88, No. 3, 1979

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Phibbs, Jr., P. V. and Bernlohr, R. W. (1971) J. Bacterial. 106: 375-385. TeGst, D. W., Meers, J. L., and Brown. C. M. (1973) The Enzymes of Glutamine Metabolism, pp. 167-182, Academic Press. Borris, D. P. and Aronson, J. N. (1969) Biochim. Biophys. Acta 191: 716-718. (1978) Biochim. J. 173: 45-52. Hemmila, I. A., and Mantsala Kane, J. F., Stenmark, S. L., Calhoun, D. H. andxnsen, R. Chem. 246: 4308-4318. A. (1971) J. Biol. Kane, J. F., Holmes, W. M.,xd Jensen, R. A. (1972) J. Biol. Chem. 247: 1587-1596. (1968) Genetics 60: 707-717. Jensen, CA. Kane, J. F., Wainscott, V. J. -i;f;d Hurt, M. A. (1979) J. Bacterial. 137: in press. Patel, N., Holmes, W. M., and Kane, J. F. (1974) J. 119: 220-227. Bacterial. Hoch, S. 0. (1914) J. Bacterial. 117: 315-317. Kane, J. F., and Jensen, R. A. (1970) J. Biol. Chem. -* 245. 2384-2390. Kane, J. F., and Jensen, R. A. (1970) Biochim. Biophys. Res. Commun. 41: 328-333. Bott, K. F, Reysset, G. and Gregoire, J. (1977) Biochim. Biophys. Res. Commun. 79: 996-1003. Dean, D. R., Hoch, J. A.,and Aronson, A. I. (1977) J. Bacterial. 131: 981-987.

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