Amino acid sequence homology among fructose-1,6-bisphosphatases

Vol. 135, No. 2, 1986

8lOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

March 13, 1986

Pages 374-381

AMINO ACID SEQUENCE HOMOLOGY AMONG FRUCTOSE-1,6-BISPHOSPHATASES Frank

Marcus,

Brigitte Gontero*, Peter and Judith Rittenhouse

B. Harrsch**,

Department of Biological Chemistry and Structure University of Health Sciences/The Chicago Medical School North Chicago, IL 60064 Received

January

1986

21,

SUMMARY: The hydrolysis of fructose 1,6-bisphosphate to fructose 6phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In the present studies, we have made a comparison of the primary structure of mammalian fructose-1,6-bisphosphatase with the sequence of peptides isolated from the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplast enzymes. Our results demonstrate a high degree of sequence homology, suggesting a comnon evolutionary origin for all fructose-1,6bisphosphatases. 8 1986 Academic Press. Inc. Fructose-1,6-bisphosphatase fructose

1,6-bisphosphate,

comparison (1)

of amino

and sheep

logy

is

regions

also

nificant

sequence

since

the

cytosolic *

(2)

evident

of muscle

an essential

acid

liver

of rabbit

region

(FbPase)'

sequences reveals

from (3)

FbPases homology

properties

liver

chloroplast

FbPase.

of

high

A

kidney degree

cortex of homo-

for

several

and from

the

NH2-terminal

however,

was to find

sig-

FbPases

(6),

and chloroplast

enzyme are

The chloroplast

pig

available

FbPase,

mammalian

from

This

information

the hydrolysis

of gluconeogenesis.

of the enzymes

More surprising,

between

of the

gluconeogenic

(4)

(5).

reaction

90% identity.

sequence

and rat

catalyzes

clearly

enzyme

distinct is

not

from

regulated

Present address: Centre de Biochimie et de Biologie CNRS, 13402 Marseille Cedex 9, France.

Moleculaire

du

Present French

Smith

and

**

1

0006-291X/86 Copyright All rights

address: Laboratories,

The abbreviations high performance

Department of Medicinal Chemistry, Swedeland, Pennsylvania 19479.

used are: FbPase, fructose-1,6-bisphosphatase; liquid chromatography.

$1.50 0 I986 by Academic Press, of reproduction in any form

Inc. reserved.

374

Kline

HPLC,

Vol.

135,

No. 2, 1986

by AMP (7,8) via

and exhibits

disulfide

coli

studies

FbPase.

Each of these

distinctive

from

FbPases.

vivo

by a cyclic

vitro

site

(12).

relieved

the

located

(13-15).

The results

sequence

information

FbPases,

show that

sequence

in spite

phosphatases

by cyclic

present

of the mammalian with

the

enzyme. results

differences degree

-In

protein unique

by AMP is some

which

FbPase include

within

structural

herein

in properties,

of the yeast

and Escherichia

Our previous presented

phos-

or chloroplast

can be aligned

of sequence

(10,ll).

enzyme from

from yeast

peptides

of a

NH*-terminus

studies,

-in

to be part

of FbPase

structural

peptides

phosphorylated

of this

yeast,

is

and chloro-

AMP-dependent

of the

by mammalian,

which

FbPase

inhibition

a characteristic

sequenced

have a high

of yeast

the

is

is presumed

11 from the

on tryptic

of significant

which

at residue

of the

in conjunction

enzyme

the

Escherichia

gluconeogenic

identification

coli,

FbPase,

has a characteristic

cerevisiae

FbPase

shared

all

prokaryotic

degradation

In Escherichia

but not

and the

structural

by P-enolpyruvate,

microorganisms

taken

the

of yeast

has permitted

enzyme

acid

for

mechanism

our comparative

eukaryotic

mammalian

event

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activation

a lower

two enzymes

above mentioned

phosphorylation

phorylation

enzyme,

AMP-dependent

mechanism

kinase

for

The Saccharomyces

plast

signalling

the

RESEARCH

We have now extended

data

cerevisiae

BIOPHYSICAL

light-dependent

(9).

to include

Saccharomyces

AND

a unique

bond reduction

structural yeast

BIOCHEMICAL

all

coli the work

demonstrate

amino (1,6) that,

fructose-1,6-bis-

homology.

MATERIALS AND METHODS: FbPase from baker's yeast (Saccharomyces cerevisiae) was purified to homogeneity by a procedure which consisted of the following steps: Extraction, heating in the presence of fructose 1,6bisphosphate, protamine sulfate treatment, ammonium sulfate precipitation, and phosphocellulose chromatography using substrate elution (12). In vitro phosphorylation of pure yeast FbPase with the catalytic subun= of beefheart CAMP-dependent protein kinase, carboxymethylation of the resulting product, and trypsin digestion of phosphorylatedS-carboxymethylated yeast FbPase was performed as previously described (12). Separation of peptides by reversed-phase HPLC was performed as described in the legend to Figure 1. Escherichia

coli

FbPase was isolated from the Escherichia coli strain Drs. J. Sedivy and D.G. Fraenkel, Hat-yard University). This r. coli strain, carrying the wild type fbp gene in the plasmid pBR322 (16), was grown aerobically for 24-48 h at 37°C in LB broth

MC1061/pJS23 (a giftof

375

Vol. 135, No. 2, 1986

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BIOPHYSICALRESEARCH

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plus ampicillin (ZOO pg/ml) and harvested by centrifugation for 15 min at 10,000 g. The pellet was then washed with a buffer (pH 6) containing 10 mM K-malonate, 0.1 mM EDTA, 5 mM MgCl and 1 mM dithiothreitol ("Buffer A"). One gram of cells was ground wi?i 2 g of alumina (Sigma type 305) in a mortar. The paste was extracted with 5 ml of buffer A and the alumina and cell debris were removed by centrifugation. The supernatant was adjusted to pH 6 with 10 mM K-malonate pH 5, and mixed with P-cellulose (Whatman P-11) equilibrated with buffer A to absorb all the FbPase activity. After 30 min of equilibration at 4°C the mixture was allowed to settle, and the supernatant decanted. The P-cellulose suspension was poured into a 0.8 cm-diameter column and washed with buffer A until the absorbance of the eluate at 280 nm was lower than 0.05. The column was then washed with buffer A without MgCl and then with a buffer (pH 6) containing 10 mM K-malonate, 0.1 mM ED?A, 1 mM dithiothreitol, and 30 mM K-phosphate. FbPase was finally eluted with buffer (pH 6) containing 10 mM K-malonate, 0.1 mM EDTA, 1 mM dithiothreitol, 20 mM K-phosphate, and 1 mM fructose 1,6-bisphosphate. The eluent front contained electrophoretically homogeneous enzyme of a specific activity of about 80 U/mg. Purified Escherichia coli fructose-1,6-bisphosphatase was carboxymethylated by the method of-stfield et al. (17), with minor modifications (18). Digestion of the S-carboxymethylated protein with trypsin (TRTPCK from Cooper/Worthington) was performed for 6 h at 37'C in 50 mM N-ethylmorpholine-acetate buffer (pH 8.5) at a 5O:l w/w ratio of FbPase to trypsin. The reaction products were separated by reversed-phase HPLC. Automated microsequencing of peptides (0.5-1.5 nmol) was performed in an Applied Biosystems 470A gas-phase protein sequencer using the standard sequencing program and the reagents provided by the manufacturer. The phenylthiohydantoin derivatives of amino acids liberated after each degradation cycle were identified and quantitated as such by HPLC using slight modifications of previously published methods (19,20). The analyses were performed with a Waters Model 840 system equipped with an IBM octadecyl column (4.6 x 250 mm). Repetitive yields were at least 90%. Amino acid analyses were performed as described by Bidlingmeyer Pep--et al. (21). tides (100-500 pmol) were hydrolyzed in vapor of 6 N HCl for 22 h at 110’. The liberated amino acids were reacted with phenylisothiocyanate and the resulting derivatives analyzed by reversed-phase HPLC on a Waters 840 system equipped with a Waters Pica-Tag column.

The HPLC analysis

RESULTS AND DISCUSSION: tryptic

digest

(Saccharomyces highly

vious

cerevisiae)

FbPase

(12),

this

phopeptide the

established

Sequence

established sequence that

data that

of residues yeast

FbPase

shown

in Fig.

on their

the site obtained

residues 1-15

for 2-16 of pig

order

derived

1A for

the

from

This

pattern

20 major

peaks

analysis

phosphorylation

of this

peptide

kidney

FbPase.

of

16 residue

NH2-terminal

is

In a pre-

sequence

purified

a

yeast

of elution.

of --in vitro

has a dissimilar 376

1A.

of at least

Peak 10 of Fig.

contained

peptides

S-carboxymethylated

presence

based

we selected

fragment

FbPase.

is

and shows the

by Roman numerals

study

because

with

phosphorylated

reproducible

designated

yeast

of --in vitro

of the

could

phosbe aligned

Additional region

work which

Vol.

135,

No. 2, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

II

,

1

I

20

40

60

1

1

I

80

1

1

,

100

WE (min) Fig. 1A. Reversed-phase HPLC of a tryptic digest of phosphorylated SThe FbPase tryptic digest was prepared as carboxymethylated yeast FbPase. described in the text. Separation of peptides was performed by reversedphase HPLC using an RP-304'column (Biokah) connected to a Spectra-Physics SP8700 solvent delivery system. The flow rate was maintained at 0.5 ml/min and the column effluent was monitored by absorbance at 214 nm. The digested sample was acidified by addition of 111 of glacial acetic acid per 40~1 of sample just before injection. The amount applied corresPeptides were eluted with a H O/ ponded to 4.5 nmol of FbPase subunit. acetonitrile gradient in a mixture of 0.07% trifluoroacetic and 0.03% The gradient ranged from 0 to 50% acetonitrile heptafluorobutyric acids. Designated peaks over a period of 120 min as shown by the broken line. were collected and lyophilized prior to E-degradation. Fig. 1B. Escherichia gradient acetonitrile

extends phosphorylation

beyond

Reversed-phase HPLC of a tryptic digest of S-carboxymethylated coli tbPase. Peptides were eluted with a H O/acetonitrile in 0.1% trifluoroacetic acid. The gradient ra$ged from 0 to over a period of 117 min as shown by the --broken line.

the site

NH2-terminus was

located

of

mammalian at

Ser-11 377

FbPases of

the

and yeast

that enzyme

the (12).

63%

unique In

Vol. 135. No. 2, 1986

TABLE Saccharomyces

BIOCHEMICAL

1.

Amino acid cerevisiae

Amino

AND

sequences of and Escherichia

Acid

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

some tryptic peptides of yeast coli fructose-1,6-bisphosphatase

Sequence

Location

in

Residue

pig

kidney

numbers

enzyme:

Homology

(%)

Yeast FbPase peptide: 7

Y-V-G-S-M-V-A-D-V-H-R R-D-S-T-E-G-F-D-T-D-I-I-T-L-P-R

15

T-F-L-Y-G-G-L-F-A-Y-P-D-D-K

255-268

16

K-L-D-V-L-G-D-E-I-F-I-N-A-M-R

72-

18

S-S-I-W-L-G-S-S-G-E-I-D-K-F-LD-H-I-G-K

314-333

30

20

L-L-Y-E-A-F-P-M-A-F-L-M-E-Q-AG-G-K

277-294

67

259-274

50

14

Y-I-G-S-L-V-A-D-F-H-R

244-254

73

15

A-G-L-V-D-I-L-G-A-Q-G-A-E-N-VQ-G-E-V-Q-

16

I-L-D-I-I-P-E-T-L-H-Q-R

16, 18,

were

FbPase

did

not

sequence

of the

peptides

aligned require

confirms

the

sequence

30

HPLC-purified

indicated

from

degree 378

of pig

amino kidney

30 to 100%. coding of amino

for

this

of deletions

of the gene high

that

for acid

1A and

tryptic

between

obtained

1) showed

sequence

58

numbering see Figs. and Methods".

data

introduction

ranging

sequence

70

homology

(Table the

the

homologies

nucleotide FbPase

within

other

structural

1A clearly

selected

easily

for

Amino acid

and 20 of Fig. five

peptides (for under "Materials

selected

to test

FbPases.

51-

40

302-313

of selected as described

we randomly

study,

of yeast

cerevisiae

91-100

G-G-I-Y-L-Y-P-S-T-A-S-H-P-D-G-K

alignments

tion

47

6

present

revealed

86

Y-I-K-F-C-Q-E-E-D-K

Each of the that

57

5

and mammalian 15,

44

FbPase

Amino acid sequences 1B) were determined

tides

-1-15

10

E. Coli pepm:

the

100

244-254

the yeast peptides

was the acid

pep-

7,

case.

sequences

FbPase.

The

or insertions A recent

and

determina-

Saccharomyces homology

between

Vol.

135,

pig

kidney

between

BIOCHEMICAL

No. 2, 1986

and yeast

the

FbPase.

two enzymes

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The overall

amino

acid

sequence

homology

is 45%*.

MANN YEAST COLI CHL MAN YEAST CULI CHL UAW YEAST COLI CHL NAM YEAST COLI CHL MAW4 YEAST COLI CHL

100 110 TEEUKNAllVEPEKRGKVVVCFOPLDGSSNIOCLV

120

130

DEEOK cl 140 SlGTIFGIVRKNSTOEPSEKOALDPGRNLVAAGVA

150

160

210 KLKKKGSIVSINEGVAKEFOPAlTEVIQRKKFPPO

220

230

MAN4 YEAST COLI CHL NAM YEAST COLI CHL NAM YEAST COLI CHL MAJW YEAST COLI CHL

250 240 NSAPVGARYVGSNVAOVHRTLVVGGIFMVPANKKS VVGSNVAOVHRTFLYGGLFAVPDDK K&

nLBST!S2:

uui nf#f=j

0 fi

PKGKLRLLVECNPWAYVNE~AGGLATTGK3?TAVLDI LLVECAPNSFIVE

AGGKGSOGHDRILDI

NAN4 YEAST CULL CHL

Amino acid sequence of tryptic peptides of yeast (Saccharomyces Fig. 2. cerevisiae) and Escherlchla co11 FbPase aligned with the amino acid sequence data of pig kidney (MAMM) and spinach chloroplast (CHL) Numbers above residues indicate their fructose-1,6-bisphosphatase. position in the sequence of pi-kidney fructose-1,6-bisphosphatase (1,6). ihe shown spinach chloroplast sequences are from reference 6; the other sequences are those reported herein, but the yeast FbPase sequence also includes the NH -terminal region (12). Amino acid residues comnon to two or more sequent E s are enclosed in boxes. Amino acids are indicated by the single-letter code, and AC- at the NH2-terminus of the mammalian enzyme indicates that it is acetylated.

2

D. Rogers,

personal

communication. 319

Vol. 135, No. 2, 1986 Having yeast

established

FbPase

test

BIOCHEMICALAND

and the mammalian

by analyzing

organism.

Escherichia

FbPase

l

from

coli

Five

jected

of these

to automated

homology

(30-73%)

the

2). tion,

described

under

of peptides the

The comparative strongly

already data

suggesting

that

from

to expand

our

from

iodoacetic

acid,

of the

peptides

derived

showing

the

of FbPase

under

digested

from the

presence

kidney

"Materials

and then

tryptic

of at least

5, 6, 14, 15,

of pig

because

the enzyme

19

and 16, were

and each determined

region

homology

a prokaryotic

overproduces

with

sequences coli

eukaryotic

as described

peak numbers

a precise

lower

an FbPase

was purified

Edman degradation

and Escherichia

sequence

aligned

peaks,

the

as the source

strain

a pattern

with

The herein cerevisiae

source

18) yielded

for

was selected

The HPLC analysis

(Fig.

peaks.

this

between

we proceeded

data

S-carboxymethylated

trypsin.

digest

enzyme,

of an E. coli

and Methods", with

homology

some structural

of the availability

(16)

sequence

BIOPHYSICALRESEARCHCOMMUNICATIONS

sequence

FbPase

subshowed

(Table

1).

for

peptides

of yeast

Saccharomyces

FbPase,

together

with

previous

spinach

chloroplast

established

sequence

shows a very

high

a common evolutionary

our

FbPase of pig

degree

(6),

kidney

for

all

on

shown

FbPase

of sequence

origin

are

data

(Fig.

conservafructose-

1,6-bisphosphatases.

ACKNOWLEDGMENTS We thank Dr. D. Rogers, The Genetics Institute, Cambridge, MA, for providing us with his data on the amino acid sequence of yeast FbPase We also thank S. Latshaw for his expert assistance prior to publication. with protein sequencing. This work was supported by grants from the National Institutes of Health (Grants AM 21167 and AM 26564), and the U.S. Department of Agriculture (Grant 83-CRCR-1-1299).

REFEREKES 1. 2. 3. 4.

I. and Heinrikson, R.L. (1982) I., Reardon, Marcus, F., Edelstein, Proc. Natl. Acad. Sci. USA 79, 7161-7165. Fisher, W.K. and Thompson, E.O.P. (1983) Aust. J. Biol. Sci. 36, 235-250. Xu, G.-F., Natalini, P., Suda, H., Tsolas, O., Dzugaj, A., Sun. S.C., Pontremoli, A. and Horecker, B.L. (1982) Arch. Biochem. Biophys. 214, 688-694. Rittenhouse, J., Chatterjee, T., Marcus, F., Reardon, I. and Heinrikson, R.L. (1983) J. Biol. Chem. 258, 7648-7652. 380

Vol. 135, No. 2, 1986

5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. ii: 21.

8lOCHEMlCALAND8lOPHYSlCALRESEARCH

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MacGregor, J.S., Hannappel, E., Xu, G.-J., Pontremoli, S. and Horecker, B.L (1982) Arch. Biochem. Biophys. 214, 652-664. Harrsch, P.B., Kim, Y., Fox, J.L. and Marcus, F. (1985) Biochem. Biophys. Res. Comnun. 133, 520-526. Preiss, J., Biggs, M.L. and Greenberg, E. (1967) J. Biol. Chem. 242, 2292-2294. Buchanan, B.B., Schurmann, P. and Kalberer, P.O. (1971) J. Biol. Chem. 246, 5952-5959. Buchanan, B.B., Wolosiuk, R.A. and Schurmann, P. (1979) Trends Biochem. Sci. 4, 93-96. Muller, D. and Holzer, H. (1981) Biochem. Biophys. Res. Comnun. 103, 926-933. Gancedo, J.M., Mazon, M.J. and Gancedo, C. (1983) J. Biol. Chem. 258, 5998-5999. Rittenhouse, J., Harrsch, P.B., Kim, J.N. and Marcus, F. (1986) J. Biol. Chem., in press. Tejwani, G.A. (1982) Adv. Enzymol. 54, 121-194. Babul, J. and Guixe, V. (1983) Arch. Biochem. Biophys. 225, 944-949. Marcus, F., Edelstein, I. and Rittenhouse, J. (1984) Biochem. Biophys. Res. Comnun. 119, 1103-1108. Sedivy, J.M., Daldal, F. and Fraenkel, D.G. (1984) J. Bacterial. 158, 1048-1053. Crestfield, A.M., Moore, S. and Stein, W.H. (1963) J. Biol. Chem. 238, 622-627. Marcus, F., Edelstein, I., Saidel, L.J., Keim, P.S. and Heinrikson, R.L. (1981) Arch. Biochem. Biophys. 209, 687-696. Tarr, G.E. (1981) Anal. Biochem. 111, 27-32. Black, S.D. and Coon, M.J. (1982) Anal. Biochem. 121, 281-285. Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L. (1985) J. Chromatogr. 336, 93-104.

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