Expression of normal and abnormal porcine kidney D-amino acid oxidases in Escherichia coli: Purification and characterization of the enzymes

Vol. 165, No. 3, 1989 December 29, 1989

EXPRESSION

BIOCHEMICAL

OF NORMAL AND ABNORMAL PORCINE KIDNEY

ESCHERICHIA Fusao Department

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1422-1427

COLI:

PURIFICATION

Watanabe,

Kiyoshi

D-AMINO

AND CHARACTERIZATION

Fukui,

Kyoko

Momoi

and Yoshihiro

of Biochemistry, National Cardiovascular Fujishiro-dai, Suita, Osaka 565, November

25,

ACID

OXIDASES

IN

OF THE ENZYMES'

Center Japan

Miyake'

Research

Institute,

1989

Expression plasmids for normal and abnormal porcine D-amino acid oxidases (E.C. 1.4.3.3, DAO) have been constructed from cloned cDNA that encodes the entire protein sequence of DAO, and the enzymes were expressed in Escherichia coli cells on a large scale. The expressed enzymes were purified to apparent homogeneity. The molecular weight of the normal DA0 (38 kD) was identical with that of DA0 purified from porcine kidney, whereas that of the abnormal DA0 was 39 kD, which comprised the normal DA0 with an additional decapeptide at its amino terminus. However, the specific activities of the two enzymes were comparable with that of natural DAO. The results indicate that the bulky decapeptide does not affect the structure necessary for the catalytic function of DA0 in the amino-terminal region. The use of a GTG triplet in the 5'-untranslated region of DA0 cDNA as the initiation codon for the synthesis of the abnormal DA0 is suggested. 0 1989 AcademicPress, 1°C.

D-Amino

acid

prosthetic

group,

polysomes D-amino

oxidase the

acids

(2).

as the

encoding

the

that

a flavoenzyme (l),

and

catalyzes

the

to understand function

amino

acid

We also

DA0

acid-substituted

(5).

the

of

this

sequence

of

established

containing

it

is

enzyme, DA0 from

we

we

activity

in

vitro

synthesis

became

of the

undetectable

porcine

mutant

cDNA clones

and human

synthesizing seven

DAOs,

and of

kidney

system single

in vitro

on replacement

of

relationship

isolated

prepared

on free

deamination

structure-function

an in vitro

Moreover,

FAD as the

synthesized

oxidative

DAOs by means of oligonucleotide-directed

of DA0 cDNA and enzymatic

biological

DAO), enzyme

enzyme

In order

(3,4).

functional

1.4.3.3,

a peroxisomal

mature

entire

cDNA libraries a

is

as

as well

(E.C.

it

for amino

mutagenesis

was found

either

Tyr-228

1 This work was supported in part by a Research Grant for Cardiovascular (62A-1) from the Ministry of Health and Welfare of Japan, Diseases 01770170 and 01770195) Grants-in-Aid for Scientific Research (63570137, the Ministry of Education, Science and Culture of Japan. 2T~

whom correspondence

Abbreviations: base pairs. 0006-291X/89 Copyright All rights

should

DAO, D-amino

acid

be addressed. oxidase;

$1.50

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

1422

SDS, sodium

dodecyl

sulfate;

that with

bp,

and from

BIOCHEMICAL

Vol. 165, No. 3, 1989 Phe or His-307 of normal

with

Leu

and unique

characterization MATERIALS

present

paper

DAOs in Escherichia

In the

coli

(6,7).

abnormal

of the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

expressed

enzymes

are

also

we report

the

cells.

expression

Purification

and

described.

AND METHODS

Materials ----Enzymes for DNA manipulation were obtained from Toyobo Co. and dehydrogenase from Oriental Yeast Co.; QAE Zetaprep Takara Shuzo Co.; lactate from Cuno Co.; and a TSK-gel G3000SW column (0.75 x 60 cm) -from Tosoh Co. Other chemicals were of guaranteed grade and commercially available. ----The competent cells, E. coli HBlOl and JM109, were Bacteria and plasmids plasmid, pKK-223-3, from obtained from Takara Shuzo Co.; and the expression Pharmacia. Preparation and manipulation of DNA ----Restriction enzymes and the competent cells, E. coli HBlOl and JM109, were used in accordance with the suppliers' recommendations. All other DNA manipulations were performed as described by Davis et al. (8). Protein sequence determination ----Amino-t.erminal sequence analysis was performed using an Applied Biosystems gas-phase sequenator, model 470A, which was connected on-line to an Applied Biosystems PTH analyzer, model 120A. DA0 assays ..-'--A henzoate-free sample of the purified DAQ was prepared by passing the D-alanine-reduced enzyme through a TSK-gel G3000SW column which had been equilibrated with 20 mM sodium phosphate buffer, pH 6.8, and then used for measurements of DA0 activity and absorption spectra. DA0 activity was assayed by coupling the DA0 and lactate dehydrogenase reactions as described (5-7). Absorption spectra were measured using a Hitachi spectrophotometer, model U-3200. Protein was determined by the method of Lowry et al. (9) with bovine serum albumin as a standard. Growth of cells for enzyme purification ----The 2 x YT medium used for the growth of E. coli cells contained 1.6 % polypeptone, 1 % yeast extract and 0.5 % NaCl. E. coli HBlOl and JM109 carrying the respective expression plasmids were grown in 12 1 of medium in a shaker incubator at 37 "C overnight, and then the cells were harvested by centrifugation at 0 "C.

RESULTS AND DISCUSSION Construction

of

a DA0 expression

kidney

DA0 efficiently

chosen

to

tat

subclone

promotor,

binding

SmaI site

the

rihosomal

of cDNA for

the

on the if

plasmid

the

DA0 protein

[SalI-PvuII

the

SalI-EcoRI

fragment

fragment

site

of pKK-223-3.

the

5'-untranslated

5'-untranslated

of

E.

rihosomal to

As the

this

region of

first which 1.34

recombinant

DNA polymerase

The resultant region

of the

This

and rrnB

fragment,

coli

DAO.

be utilized of

plasmid, the cDNA,

cDNA. Bal 1423

order

to

expression

site

DA0 (3), of

for

codon

site. kidney

In

can

initiation

binding porcine

-----

prokaryotic

cDNA fragment

the

Klenow

E. coli,

a multicloning

site

the

in

system

the

insert

contains

within

I and

coding into

then

subcloned still

next

31 exonuclease

step, digestion

fragment

pUC19. into

contained to

into

15 hp of region

was blunt-ended

therefore, As the

cloned

enzyme

whole

was subcloned plasmid

a strong

10 to

a restriction the

was

The rihosome

an insert

is

porcine

pKK223-3

terminators.

contained kh],

vector

plasmid

express

step,

express

of Then

with

the

the

SmaI

68 hp of remove of

the

the cDNA

Vol. 165, No. 3, 1989

BIOCHEMICAL

fragment was carried out, n i z A was again subcloned

and then

resultant

plasmid,

of the

cDNA, which

the

insert.

the

productivity

in

place

fragment

of

the

DA0 protein,

pBR322

sequence

digestion

Bal

intact.

expression (pw-DAOL),

only

2 bp of the

Another

around

the was

into

the

The

the

coli

plasmid,

plasmid above. the

was also

However,

the

5'-untranslated

as

HBlOl

of and

SphI-ScaI

resultant

expression 68 bp of

5'-end

plasmid

was introduced

subcloning

designated

E.

The region

of the

sequence

as described

leaving plasmid

by

above.

5'-untranslated

of pUC19.

1.

same procedure

transferred

the

pUC19 vector

sites

an appropriate as

copy number

pKK223-3

in Fig.

to

vector

sequencing

increase the

off

pUK-DAOL.

(pUK-DAO)

These or

JM109

respectively. of

introduced

into

toward

rabbit

of

proteins.

DA0 proteins E. coli

in

cells

anti-porcine

was

with

protein

also

gave

to about

39 kD,

in

compared

to

cells the

those the

of

from

kidney

that

the

pUK-DA0

2 shows gave

from

and

a band

of

protein

the

blotting which

kidney

protein at

productivity

38 kD. of the

27

1. Schematic diagram of expression plasmid pm-DA0 for porcine kidney DAO. The closed and open boxes represent the coding and untranslated regions of the cDNA for DAO, respectively. The closed and open arrows correspond to the tat promotor and rrnB terminators, respectively. Region a was derived from cDNA for DAO, pDAO-10 (3); region b, pKK-223-3; region c. pUC 19.

Fig.

coli b,

cells porcine

1424

expressing kidney

a, DAO. homogenate

from

corresponding

(ScaVSmal)

Fig. 2. Western blotting of E. from porcine kidney (100 ng); pUK-DAOL; d, pUK-DAO; e, pKK-DAO.

the

and the

derived

band was at a position that

pUK-DAOL proteins

Western

porcine

The

plJK-DAO-derived

indicating

pUK-DA0

of immunoreactive Fig.

homogenate.

of the low,

Both

synthesis

DA0 purified

However,

was very

-----

DA0 antiserum. derived

one band.

of pKK-DA0

coli

directed

protein

identical

pUK-DAOL

E.

kidney

The

immunoreactive

expression

expression

of

expression were

same

SphI-SmaI

shown the

This

Expression

mobility

to

31 was omitted,

vectors

the

the is

by essentially with

region

step,

into

as pUK-DAO,

trimmed

by nucleotide

final

the

fragment

contained

was confirmed

of pKK-DA0

constructed

the

As the of

designated

the

into

pKK-DAO,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

purified (250

enzyme pg>; c,

The DA0

BIOCHEMICAL

Vol. 165, No. 3, 1989 protein

increased

by more

pUC19.

The expression pUK-DA0

of the

600 ml of sodium

benzoate.

to the

The

supernatant,

precipitate

was

dissolved

in

the

collected

1 mM sodium sample

proteins

were

phosphate

TSK-gel

buffer,

at

peak

fractions

purified

DAO.

66 g of E. coli

A containing at

low

Properties

of the

those

In

on

with weight

consistent

with

8.31

comparable

8.98 that

from

Amino-terminal

umol of

kidney

2.

(Fig.

by The

and

then

1 mM sodium pH 8.0,

and

then

the

had been equilibrated and

then

M KCl.

the

The

The

absorbed

eluate

was

precipitate

was

volume

of

20 mM

chromatographed

a Hitachi

was

high

fractionated

observed

on

a

performance by monitoring

for

the

collected,

eluate.

The

and then

used

21 mg of DAO-N was obtained of DAO-A was less

The purified

than

preparations

that

kidney

and

system The

as from

of DAO-N

(2,3). to

were

vitro

39 per

also

other The

of The

identical

The DA0

hand,

the were

DAO-N and DAO-A which

absorption

as well

to with

results

mg protein,

(5). states

3).

identical synthesized

kD.

activities

kidney

was

On the

be

oxidized/min from

in

judged

(Fig.

which

the

specific

4)

were

electrophoresis to be 38 kD,

and D-alanine-reduced

as the with

were spectra

oxidized

those

of

DA0

(10).

sequences fragment

using

estimated

D-alanine

benzoate

of DAO-N and DAO-A are SalI-PvuII

Fig.

min)

24 hr,

then

were

1 mM

JMlOQ.

lysate was

DA0 purified

oxidized with

porcine

30

a minimum

was

polyacrylamide-gel

DAO-A in

in

were

yield

DAOs -----

from

of

and

DAO-N complexed

the

in E. coli

SDS

A, 0.2

preparation,

However,

those

the

peaks

in

g) was added

20 mM Tris-HCl,

Buffer

DA0 activity

a typical

g,

that

eluate

of DAO-N was estimated

with

of DAO-N in

showed

a reticulocyte

molecular

purified

that

x

column

sample

The

635A.

DA0 purified

obtained

same buffer,

purified

weight of

the

Several

expression

homogeneous

molecular

The

derived

precipitated (136

0 "C for

dissolved

pH 6.8.

nm.

then

50 % saturation.

then

model 254

HBlOl.

due to the

were

Buffer

with

chromatograph, absorbance

with

sulfate and

G3000SW column

liquid

be

with

ammonium

washed

that

respectively.

40 uM FAD and

at

than

at 0 "C overnight.

against

A)

to

40 uM FAD and

and

containing

less proteins

sulfate

(5,000

dialysed

insert

(66 g) was suspended

sonicated ammonium

(Buffer

was

paste containing

to a QAE Zetaprep

by centrifugation

sodium

major

was

column

the

was incubated

pH 8.0,

benzoate

eluted with

collected

the

The

was Solid

mixture

solution

was applied

A.

fractionated

the

sample

cell

centrifugation

20 mM Tris-HCl,

containing Buffer

by

was also

Hereafter,

pH 8.3,

suspension

and then

of the

to as DAO-N and DAO-A,

buffer, 20 min).

ligation

protein

DAOs -----The

cell

The

with

referred

x g,

benzoate. dialysed

are

expressed

(5,000

final

to some extent.

50 mM pyrophosphate

centrifugation

on the

pUK-DAOL-derived

protein

and pUK-DAOL

Purification

lo-fold

of the

of the pUK-DAO-derived from

than

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of DAO-N and DAO-A ----shown

in Fig.

of DA0 cDNA is

5. also

A part shown.

1425

The amino-terminal the

nucleotide

The

amino-terminal

sequences

sequence

of the

sequence

of

Vol. 165, No. 3, 1989

a

94-

BIOCHEMICAL

b

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cdefg

1*

67-

30- 4m

03

0 4

MIX lO-3

300

400 WAVELENGTH

500 (nm )

f

Fig. 3. SDS-polyacrylamide gel electrophoresis of the purified DAO-N and DAO-A synthesized in E. coli. a and g, molecular weight standards; b, the purified DAO-N (13 ng); c, the purified DAO-A (2 ng); d-f, porcine kidney DA0 (d, 2 ng; e, 4 ug; f, B vd. Absorption spectra of DAO-N purified from E. coli. (A) the oxidized E?i B) the D-alanine-reduced form, (C) the oxidized form in the presence of 1 mM benzoate. the

first

from

14 amino

DA0 cDNA (3)

hand,

the

DAO-N.

an

underlying

are

the

the

the

two

first

significant

of

in

identical

(166-168)

was

acid with

assumed

is

the

to

amino

often in

of

be the

initiation

Fig.

5 was

codon

ACA

ACC T T G Ai?GGA

CCA CAG GCT GGC ACG &

(B) (9

+hr-Thr-Lcu-Lys

Fig.

5.

-Gly-

Pro-G,“-

Ala-

Glyahd-

an

of

- Val - Val-

i&Arg-Val

- Val -Val

Ile-

-k

mechanism

the

in

Fig.

DA0 cDNA

by assuming

DAO-A. when

Gly -Ala-

- Giy -Ala-

no

However,

the

second

DAO-A

AGT TCA C’!:

:AG

region a FAD binding

site

(13)

in

DAO-N

is

indicated

1426

by

a double

underline.

a

GTG

might

AGT CTG%G

be

CAG

GGC GTC A T T GGG CTG TCC Gly-Val

Gly-

- ik-Giy-

Vd -k-

21

LCU-%I

Gly-

LCU

amino-terminal sequences of DAO-N and DAO-A. (A) A part of the sequence of the cDNA for DA0 (3), (B) the amino acid sequence of (C) the amino acid sequence of DAO-A. GTG codons in the 5'-untranslated A sequence characteristic of of the cDNA are denoted by closed circles. The

in

However,

shift.

nucleotide DAO-N,

the

comprises

analysed

Therefore,

d.?A

of

with The

CGT GTG GTG GTG A T T GGA%A

&Arg-Val

that

codon

for

obtained

other

initiation

due to a frame

TC GAC AGA f%

from DAO-A

region

codon.

(A)

On the

As can be seen as

was

predicted

identical that

used

initiation DAO-A

that

terminus.

analysed.

was obtained

that

was

indicate

its

sequence

sequence

(11).

5 '-untranslated

As GTG is nucleotide

kidney

Met(l1)

at the

with

of DAO-A differed

results

of DAO-A was thus

GTG (149-151)

sequence

acids

The

decapeptide

agreement

from

following

DAD-N.

GTG triplets the

amino

10 amino

sequence

circles).

(12),

DA0 purified

first

expression

(solid

prokaryotes

of

additional

the

there

that

sequence

DAO-N with

that

of

However,

fragment

of DAO-N was in complete

and

sequence

amino-terminal

5,

acids

Vol. 165, No. 3, 1989

expressed as the

in E. coli initiation of

for

only

six

It

the is

would

amino

catalytic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GTG triplet

site

acid

residues would

sequence

with

from

the

166-168

presence

amino-terminal

this

interfere

function

that

in the If

not

at positions

interesting

underline).

decapeptide

the

with

a FAD binding

(13)(double

the

being

cells codon.

characteristic suggested FAD,

BIOCHEMICAL

the

the

region

is

the

binding

site,

even

of DAO-N is

FAD,

site in

structure

when

the

used

a sequence

binding

of

and the

be maintained

of

being

for

spite

of

necessary

decapeptide

is

attached. In this

paper,

transformed of

E. coli

cDNA encoding

Therefore,

it

encoding

cells. the

of

DA0 protein the

be possible

enzyme through

the

present to

elucidate

expression

the

The

entire

is much simpler

a partial

availability should

we described

expression

expression

system

DA0 protein than

the

of normal

sequence complicated

sequence expression

the

procedure

purification

structure-function

of mutant

characterized as

reported and

the

is

and abnormal

by the

starting

DA0 cDNA

(14). system

relationship

use

material.

involving

recently

DAOs in

With for

DAO, of

the it this

enzymes.

REFERENCES 1. de Duve, C. and Baukhuin, P. (1966) Physiol. Rev. 46, 323-357. 2. Fukui, K., Momoi, K., Watanabe, F. and Miyake, Y. (1986) Biochem. Biophys. Res. Commun. 141, 1222-1228. 3. Fukui, K., Watanabe, F., Shibata, T. and Miyake, Y. (1987) Biochemistry 26, 3612-3618. 4. Momoi, K., Fukui, K., Watanabe, F. and Miyake, Y. (1988) FEBS Lett. 238, 180-184. 5. Fukui, K., Momoi, K., Watanabe, F. and Miyake, Y. (1988) Biochemistry 27, 6693-6697. 6. Watanabe, F., Fukui, K., Momoi, K. and Miyake Y. (1988) FEBS Lett. 238, 269-272. 7. Watanabe, F., Fukui, K., Momoi, K. and Miyake, Y. (1989) J. Biochem. 105, 1024-1029. M. D. and Battey, J. F. (1986) Basic Methods in 8. Davis, L. G., Dibner, Molecular Biology, Elsevier, New York, Amsterdam, London. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 10. Yagi, K. and Ozawa, T. (1962) Biochim. Biophys. Acta 56, 420-426. 11. Ronchi, S., Minchiotti, L., Galliano, M., Curti, B., Swenson, R. P., Williams, C. H., Jr. and Massey, V. (1982) J. Biol. Chem. 257, 8824-8834. 12. Kozak, M. (1983) Microbial. Rev. 47, l-45. 13. Schulz, G. E., Schirmer, R. H. and Pai, E. F. (1982) J. Mol. Biol. 160, 287-308. 14. Ciccarelli, E., Massaer, M., Guillaume, J. -P., Herzog, A., Loriau, R., Cravador, A., Jacobs, P. and Bollen, A. (1989) Biochem. Biophys. Res. Commun. 161, 865-872. 161, 865-872.

1427