Requirement of GTP for pteridine synthesis in Salmonellatyphimurium and its inhibition by AMP

BIOCHEMICAL

Vol. 20, No. 4,196s

REQUIREMENT SYNTHESiS

AND BIOPHYSICAL

OF GTP FOR PTERIDINE

IN SALMONELLA

TYPHIMURIUM

From R. Dalal Department

RESEARCH COMMUNICATIONS

AND

ITS INHIBITION

BY AMP*

and Joseph S. Gots

of Microbiology, School of Medicine, University Philadelphia, Pennsylvania 19104

of Pennsylvania

Received July 14, 1965

We hove been studying

a number of mutants of Salmonella

is acutely inhibited

whose growth

by adenine.

and the types of agents that can relieve impairs

growth

biosynthetic

by decreasing

reactions.

acid biosynthesis inhibitors.

the inhibition

the wailability

1964) and the condensation dihydropteroic

derivative

of reduced

acid (Brown,

units for various

effect of adenine

to a pteridine

to a pteridine

communication)

is inhibited

using lactobacillus

showed that GTP is specifically

guanine

by adenine

as

and Brown,

with pABA to form

for pteridine

Supported by grants from the U.S. Public National Science Foundation (68-2924).

509

congener

ribonucleotides.

and Levenberg required

derivatives

1959).

The results show that the participating conversion

of folic

those required

(Reynolds

hydroxymethylpteridine

1959; Shiota,

that adenine

have led us into an analysis

and the possible

of a guanine

obtained

has suggested

This paper deals with the first enzymes to be studied,

for the conversion

*

patterns

of single carbon

These considerations

in Salmonella

The sowth

typhimurium

Health

(1965) with

is GTP and that its Shiota

(personal

Pseudomonas

also

formation.

Service

(CA-02790)

and the

Vol. 20, No. 4,19&T

BIOCHEMICAL

AND BlOPHYSlCAL

MATERIALS

The preparations and Brown (1964). some adenine Cultures

sensitive

were gown

mutants (ads-) derived

The cells were harvested,

sonic disintegrator

were patterned

used were Salmonella

in a minimal

(0.03 M at pli 7.2),

AND METHODS

and experiments

The bacteria

RESEARCH COMMUNICATIONS

typhimurium,

from it (obtained

salts-glucose

washed twice

after the work of Reynolds strain LT-2

from M.

and

Demerec).

medium for 18 hours with aeration.

with saline,

containing

2-mercaptoethanol

and centrifuged

at 30,008

suspended (4 mM),

in phosphate

ruptured

Xg for 1 hour.

buffer

by a MSE ultra-

The clear extract

was

stirred with 5% acid washed Darco grade 20 x 40 for 20 minutes at 4 C and recentrifuged to remove the charcoal. overnight

The charcoal

at 4 C, each time against

2-mercaptoethanol

I) were carried

Folic acid compounds

assay with Streptococcus

faecalis

Activity

The folate

was expressed

compound

of the reaction

(Usdin -0 et al.,

out at 37 C under paraffin were determined

in terms of mug of folate

formed was identified mixture

on Whotman

No.3

and purified

chemically

equivalents

acid as formed.

chromatography

MM paper using phosphate

solvent and then visualized

by tetrazolium

bio-

1954) with S. faecalis.

from pteroylglutamic

by paper chromatography

The hydroxymethylpteridine

oil in

and folic

by paper ascending

Reduced 2-amino-4-hydroxy-6hydroxymethylpteridine was prepared

twice,

by microbiological

(ATCC 8043) as the test organism

buffer (0.1 M at pH 8.0) as developing autography

was then dialyzed

fresh tris buffer (0.03 M at pH 8.0) containing

11 x 100 mm test tubes.

of an aliquot

extract

(4 mM).

Enzyme studies (see Table

the standard.

treated

acid by the method of Wallerzd.

to remove all traces of pteroylglutamic

was reduced

acid for 30 minutes and adjusting

(hydroxymethylpteridine)

just before use with sodium borohydride

to pH 8.0.

510

(1950) acid. and

BIOCHEMICAL

Vol. 20, No. 4,1965

AND BIOPHYSICAL

RESULTS AND

Synthesis requirements MgCl2

as the Escherichia

and reduced

times the optimal adenine

level did not inhibit

detectable

to be due to inhibition

synthesis.

with guanosine The low activity

synthesis, by ATP (Table

Two Step Conversion

Additions

to reaction

Stage 1 A B C D E F G H I

GTP GTP + ATP GTP +ATP -I- pABA ATP ATP + pABA -

showed the same viz,

ATP,

in ATP concentration

Previous growth

type strain and the adenine

Table

System

Increase

nor was there any significant

the hydroxymethylpteridine

GTP allowed

of Salmonella

-- al.J1961), -coli system of Brown et

hydroxymethylpteridine.

of the wild

DISCUSSION

acid by extracts

did not repress the activity,

the activities replace

of dihydropteroic

RESEARCH COMMUNICATIONS

sensitive

pABA, to four

in the presence difference

mutants.

of

between

Attempts

or GMP were unsuccessful, with GTP eventually

to

but

was found

Ill). I

of GTP to Dihydropteroate

mixture

Total folate

in

equivalents (mlrd

formed

Stage 2 ATP + pABA + HMPt* ATP -I- pABA pABA GTP + ATP + pABA ATP + pABA PA@ ATP + p4BA

69. I 42.2 14.7 16.6 5.1 0 0 0 0

The reaction mixture contained: MgCl2, 2.5 mM; potassium phosphate, 10 mM; CuCl2, 5 x 104 mM; sodium ascorbate, 5 mM; and 2-mercaptoethano1, 5 mM in a total volume of 1 ml of 0.1 M tris buffer, pH 8.0. Incubation for the first stage was carried out for 2 hours with 0.2 ml cell-free extract (3.2 mg protein) and the reaction ws stopped by heating to loo0 for 1 minute. After cooling incubation for the second stage was carried out with 0.1 ml of fresh cell-free extract (1.6 mg protein) for a further 2 hours. GTP, 0.4 mM; reduced hyckxymethylpteridine (HMPt+), 0.4 mM; ATP, 1 mM, and pABA, 4 mM, were added as indicated above. 0.2 m moles of Pmercaptoethanol vms added and the reaction was stopped by immersing the tubes in a boiling water bath for 1 minute.

511

Vol.

20,

No.

41965

BIOCHEMICAL

In order necessary

to split

shown

that

GTP

Incubation in the

not out

of GTP

conversion

obtained

ATP

inhibits

II.

folate

activity

the

GTP

via

ATP

is added

conversion GTP

results

about

16% of ATP,

efficient

inhibitor

when

either

of the

activity

AMP

since

Guanine

of Guanine

(Table rather

than

could

result

only

be converted

enzyme. first

or ATP.

The

stage

to

eight

fold

I,

B vs E)

(fable

during

this

B vs C). guanosine,

GMP

borne

and

out

Brown

by data

(1964)

or GMP

or GDP

is the

presented

in E.coli,

is substituted

for

most in

negligible GTP,

and

Ill.

AMP

of GTP. adenosine

three.

as inhibitors

It is unlikely

chromatographic

is seen

that

analysis

AMP showed

in Table

acts that

by depleting appreciable

II to Form

Hydroxymethylpteridine

Folate

compound

None

equivalents farmed (mpg/mg protein)

0

Guanosine GMP GDP GTP The reaction was carried of the guanine compound

then

had

pABA

presumably

fresh the

I,

guanosine

and

Compounds

of either

to hydroxymethylpteridine

Table Ability

during

by Reynolds

of the

transphosphorylation

and

is further

obtained

experiments

therefore which

converted

that

is observable only

most

being

absence

should

of pABA,

COMMUNICATIONS

it was

Preliminary

in the

of hydroxymethylpteridine,

Unlike

stages.

of pABA

GTP

this

observation

A comparison is the

when

that

Table

two

to pteroate

addition

is indeed

initial

into

RESEARCH

to hydroxymethylpteridine

to hydroxymethylpteridine

GTP

precursor

gives

reaction

that

The

BIOPHYSICAL

of GTP

in the absence

subsequent

and

conversion

converted

by the

indicates

active

the

overall

was

conversion

higher

GDP

the

carried

pteroate

stage

to study

AND

0 0.36 2.00 12.80 out as in Table I system B. GTP was substituted indicated which were added in Stage 1.

512

by

0.4

mM

BIOCHEMICAL

Vol. 20, No. 4,1965

amounts of GTP remained hibited

to a slightly

relatively

AND BIOPHYSICAL

greater

extent

than the wild

(Table

Adenine

compounds

mutants are in-

during

growth

of

of the mutants does

Ill). Table

of Formation

sensitive

type strain LT-2 and the presence

(50 pg/ m I) o f a d enine

high concentrations

not repress enzyme activity

Inhibition

The adenine

after the reaction.

RESEARCH COMMUNICATIONS

III

of Hydroxymethylpteridine

from GTP by Adenine

Compounds

Foiate equivalents formed (mpg/mg protein in stage 1)

added

Stage 1

Stage 2

LT-2

ATP AMP Adenosine

ATP AMP Adenosine -

12.4 6.8 9.2 3.8 14.4 13.2

ads-2 G7b 14.4 15.0 5.2 4.6 9.6 8.8 1.7 2.1 13.0 11.0 12..7 11.7

ads-4 K-Y?0 8:7 9:1 10.1 12.1 2.4 2.5 16.8 16.0 14.9 16.0

The reaction mixture was as in Table I. GTP, 0.4 mM was added in stage 1; ATP, 1 mM and pABA, 4 mM in stage 2. The adenine compounds, 1 mM were added as indicated. For adenine sensitive mutants: (a) indicates cells grown without supplementation, ,(b) indicates cells grown In medium containing adenine (50 pg/mI) and thiamineQ &ml) to permit growth, Table IV Inhibition

AMP

concentration stage 1

of Formation

of Hydroxymethylpteridine Concentrations of AMP

in

from GTP by Different

Folate equivolents formed (mpg/mg protein in stage 1) LT-2 647) 12.1 10.2 8.6 6.8 5.1 5.5 2;a 3.2 1.6 1.5

0

0.2 O-5 1.0

2.0

ads-4 TT 13.1 6.2 3.6 1.8 0.8

ads-5 (al-04 12.4 9.9 6.6 3.4 0.9

12.5 9.4 5.2 3.3 0.9

The reaction mixture was as in Table Ill. AMP was added in stdge 1 as indicated, and in stage 2 to give a final concentration of 2.0 mM in all cases. For adenine sensitive mutants, (a) and (b) are as in Table III. For LT-2: (a) indicates cells grown without supplementation (b) indicates cells grown in 200 &ml adenine.

513

Vol. 20, No. 4,1965

The inhibition Table

IV.

the wild wild

The activity

BKXHEMICAL

produced

by increasing

of the adenine

entirely

concentrations

sensitive

type at almost all AMP concentrations.

type strain at high concentrations

inhibition,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

it is unlikely on this basis.

of adenine

that the acute sensitivity Other

reactions

mutant (ads-4) Though

of AMP

can be seen in

is greater

inhibition

than that of

of growth

of’ the

may be due to this type of enzyme of the mutants can be explained

of folic acid metabolism

me being

investigated

for this purpose.

REFERENCES Brown, G.M., Fed.Proc.c,19 (1959). Brown, G. M., Weisman, R,A, and Molnar, D.A., J.Biol.Chem.236,2534 (1961). Lever&erg, B., Fed. Proc.24,669 (1965). Reynolds, J.J. and &own,-. M., J.Biol.Chem.239,317 (1964). Shiota, T., Arch.Biochem. Biophys.~,155 (1959).Usdin, E., Shockman, G,D, and Toennies, G., Appl.Microbiol.2,29 (1954). Waller, C.W., Goldman, A.A., Angier, R.B., Boothe, J.H., Hutchings, B.L., Mowat, J.H. and Semb, J., J.Am.Chem.Soc.z,4630 (1950).

514