Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase

Vol. 133,No.

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS Pages 520-526

2, 1985

December17.1985

AMINO ACID SEQUENCE SIMILARITY BETWEEN SPINACH CHLOROPLAST AND MAMMALIAN GLUCONEOGENIC FRUCTOSE-1,6-BISPHOSPHATASE Peter

Yangkil

B. Harrsch*,

Kim?

J.

Lawrence

Fox**and

Frank

Marcus*

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

Received

October

28,

of Molecular Biology, Abbott Park, Illinois

Abbott 60064

Laboratories

1985

SUMMARY: Chloroplast fructose-1,6-bisphosphatase is an essential enzyme inphotosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, Scarboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversedphase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose-1,6bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation. B 1985 Academic Press. Inc.

Fructose-1,6-bisphosphatase the hydrolysis been isolated genie

of fructose

1,6-bisphosphate

from a variety

tissues,

enzymes

(E.C.3.1.3.11),

yeasts

are very

fructose-1,6-bisphosphatase However, contain

as with

Copyright All rights

that

other

enzymes

used are: acid.

involved

Inc. reserved.

520

also

cytoplasm

pair

(4).

performance

these

extend

of leaves

Chloroplast

to

(3).

metabolism,

liquid

has

gluconeo-

of all

in carbohydrate

isozyme

$1.50

0 1985 by Academic Press, of reproduction in any form

marrmalian

similarities

in the

HPLC, high

include

catalyzes

6-phosphate,

The properties

and these

present

a chloroplast/cytoplasmic

The abbreviations TFA, trifluoroacetic 0006-291X/85

of sources

(1,2)

enzyme that

to fructose

and microorganisms.

similar

the

plants fructose-

chromatography;

Vol. 133, No. 2, 1985

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

1,6-bisphosphatase

is

essential

in the

dioxide

fixation

into

sugars

(5,6)

clearly

distinct

from

the cytosolic

tase.

The chloroplast

enzyme

unique

light-dependent

activation

system

in which

a critical

groups

(9,lO).

Despite

similarity tases

between in their

the amino

acid

phosphatases were

mammalian plast

all

homology

tases,

suggesting

lyzing

the

mechanism

a possible

mammalian

a common

same reaction

with

but

regulated

appears

(11).

present

relationship

Since

demonstrate

studies between

and the

origin

to be some

fructose-1,6-bisthe

and chloroplast

evolutionary

sulfhydryl

subtilisin

(12,13),

studies

a

fructose-1,6-bisphospha-

structural

of these

to free

there

fructose-1,6-bisphosphatase

The results between

thioredoxin

gluconeogenic

established

enzyme are

a ferredoxin/

and gluconeogenic

of two mammalian

of this

and exhibits

however,

sequence

of carbon

by AMP (7,8)

reduced

digestion

pathway

fructose-1,6-bisphospha-

via

bond is

to limited

gluconeogenic

quence

not inhibited

response

for

properties

gluconeogenic

differences,

chloroplast

to probe

enzyme.

and the

disulfide

has been already

designed

is

photosynthetic

spinach

chloro-

significant

se-

fructose-1,6-bisphosphafor

by completely

these

enzymes

different

cata-

mechanisms.

MATERIALS AND METHODS: Spinach chloroplast fructose-1,6-bisphosphatase was purified to homogeneity by a method based on previously published procedures (14,15). The improved method consists of extraction, ammonium sulfate fractionation, Sephadex G-100 gel filtration, DEAE-cellulose ion exchange chromatography and hydroxylapatite chromatography. Details of the purification will be published elsewhereI. The purified enzyme was and we have confirmed its subunit electrophoretically homogeneous, molecular weight of 45,000 (cf. Fig. 2A, ref. 16) and a native molecular weight of 180,000 (cf. Fig. 2, ref. 17). The techniques used for S-carboxymethylation and protein cleavage with cyanogen bromide were those previously described (18). Digestion of the S-carboxymethylated protein with trypsin (TRTPCK from Worthington) was performed for 24 h at 22" in 100 mM N-ethylmorpholine-acetate (pH 8.5) at a w/w ratio of fructose-1,6Separation of peptides by reversedbisphosphatase to trypsin of 1OO:l. phase HPLC was performed as described in the legend to Figure 1. Automated microsequencing of peptides (0.5-1.5 nmol) was performed in The phenylthiohyan Applied Biosystems 470A gas-phase protein sequencer. dantoin derivatives of amino acids liberated after each degradation cycle were identified and quantitated as such by HPLC using slight modifications The analyses were performed with of previously published methods (19,20).

'P-B.

Harrsch

and F. Marcus,

in preparation. 521

Vol.

133,

BIOCHEMICAL

No. 2, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

a Waters Model 840 system equipped with an IBM octadecyl column (4.6 x 250 Repetitive mm) or a Beckman Model 345 system with an Altex PTH column. yields were at least 90%. Amino acid analyses were performed as described by Bidlingmeyer Peptides (100-500 pmol) were hydrolyzed in --et al. (21). The liberated amino acids were reacted vapor of 6N HCl for 22 h at 110’. with phenylisothiocyanate and the resulting derivatives analyzed by rever;Ey;;;ase HPLC on a Waters 840 system equipped with a Waters Pica-Tag . RESULTS AND DISCUSSION: phatase

spinach

was S-carboxymethylated

gen bromide

by reversed-phase

(Fig.

1A) showed

numerals

based

sequence

degree

presence

on their analysis

iodoacetic

order

this

tose-1,6-bisphosphatase ment peak V for

peaks

Automated

with

products

were

The elution

designated

sepa-

pattern

1A was selected

exhibited

the highest

assay")-positive to denatured

crossrat

Edman degradation

sequence

cyano-

by Roman

Peak V of Fig.

antibody

gave the

cleaved

reaction

imnunosorbent

(18).

35 cycles

acid,

CNBr fragment

a specific

fructose-1,6-bisphos-

1A legend).

of elution.

because

against

Fig.

of 7 major

("enzyme-linked

(22)

chloroplast

and the

HPLC (see

the

of ELISA

reactivity

with

(4 mg/mg of protein),

rated

for

Purified

liver

fruc-

of CNBr frag-

Ser-Phe-Ile-Val-Glu-Gln-Ala-

Gly-Gly-Lys-Gly-Ser-Asp-Gly-His-Gln-Arg-Ile-Leu-Asp-Ile-Gln-Pro-Thr-GluIle-His-Gln-Arg-Val-Pro-Leu-Tyr-Ile-Gly. A computer established

sequence

revealed aligned

search

that with

homology.

the

Since blocked

kidney

(Fig.

of residues

285-319

The alignment

or insertions

and revealed

spinach

at the

the alignment

NH2-terminus,

between

was sought

by performing

S-carboxymethylated

above sequence (12)

kidney

require high acid

analysis

with

degree

of sequence

identity

and 20% of

appeared regard

to be

to sequence

fructose-1,6-bisphosphatase on tryptic

fructose-1,6-bisphosphatase. 522

introduction

similarity.

evidence

and chloroplast

fructose-1,6-

the

fructose-1,6-bisphosphatase

sequence

chloroplast

not

a very

66% overall

additional

mammalian

did

and the

CNBr peptide

of pig

shows 46% amino

giving

chloroplast

similarity

the

fructose-1,6-bisphosphatase

2).

replacements

between

fructose-1,6-bisphosphatase

sequence

Indeed,

conservative

of pig

homology

the chloroplast

bisphosphatase of deletions

to find

peptides

from The tryptic

Vol.

133,

No. 2, 1985

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

III

II I

LA lb

.

io

jo

40 TIME (min)

50

60

23

TIME hinl

F

Reversed phase HPLC of the fragments produced by cyanogen romide cleavage of S-carbox eth ated spinach chloro ast ructose-1,6il;phosphafase. The peptide:ix&e (2 5 1) ' lOO';l off6M guanidinewas inJeCted into a Bio Rad RP-304 ;ol:zi (41: x 250 mm) equilibrated with 0.1% TFA. Peptides were eluted with a H O/acetonitrile gradient containing 0.1% TFA (0 to 72% acetonitrile ov a r a period of 70 min) and a flow rate of 0.75 ml/min. The profile shows absorbance (A) at 214 nm (full scale = 1.0 A). Major peaks in the chromatogram are designated by Roman numerals based on their order of elution. Fig. 1B. Reversed phase HPLC of a tryptic digest of S-carboxymethylated spinach chloroplast fructose-1,6-bisphosphatase. The tryptic digest was adjusted to pH 6 with 50% TFA and 50 ,~l were injected into a Bio Rad RP-304 column (4.6 x 250 mm) equilibrated with 0.1% TFA. Peptides were eluted with a H O/acetonitrile gradient containing 0.1% TFA (0 to 63% acetonitrile ov 6 r a period of 110 min) and a flow rate of 0.5 ml/min. The peaks were numbered based on their order of elution, but only the numbers of the sequenced peptides are shown. In the text, these tryptic peptides are designated T- followed by the indicated Arabic numeral.

peptides showing peaks, for

were the

separated presence

designated sequence

contained cysteine

of in

Fig.

residues;

reversed-phase

at

least

27

1B as

peaks

Prior

analysis. a single

by

peptide. while

amino Two

the

third

of

HPLC peaks

(Fig.

number acid the

one,

523

which 1B). 16,

analysis

21,

a pattern

Three and

23,

indicated

peptides, T-23,

yielded

T-16 was

devoid

of

these

were that

and

T-21, of

cysteine

selected each

peak

contained resi-

Vol.

133.

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

No. 2, 1985

20

MAW4

Ac:DfJAAFDT:i"TLTRF"NEQGRKAR

CHL 30 40 GTGEMTQLLNSLCTAVKAISTAVRKAGIA"LvG16:

MAHM CHL Mm4 CHL

50

GSTNVTGDQ7iKKLDVLSND%INVLKSSFgiTCVLV li&~EVFSSC~R

100

110

120

130

TEEDKNAIIVEPEKRGKVVVCFDPLDGSSNIDCLV

140 SIGTIFGIVRKNSTDEPSEKDALQPGRNLVAAGYA

150

160

210 KLKKKGSlVSINEGVAKEFDPAITEVlQRKKFPPD

220

230

240 250 NSAPVGARVVGSMVADVHRTLVVGGIFMVPANKKS

260

270

330

Fig. 2. Amino acid sequence of pig (upper line) aligned with sequenced

kidney fructose-1,6-bisphosphatase peptides of spinach chloroplast fructose-1,6-bisphosphatase (lower line). Numbers above residues indicate their position in the established sequence of pig kidney fructose-1,6-bisTwo corrections to our previous sequence data are phosphatase (12). included: Gly is shown at position 156 instead of Glu; and Gin-228 replaces Glu. The sequence of the pig kidney enzyme is taken as typical of mammalian (MAMM) fructose-1,6-bisphosphatases. The only other known mammalian fructose-1,6-bisphosphatase sequence, that of the sheep liver enzyme (13). shows only 32 changes in 335 residues. The shown spinach chloroplast fructose-1,6-bisphosphatase sequences (CHL) are those reported herein. Identical amino acid residues in both sequences are shown enclosed in boxes. Amino acids are indicated by the single-letter code and AC- at the NH2-terminus of the marmnalian enzyme indicates that it is acetylated.

dues.

The

peptides

other was

medium

sized

ture

of

tion

and

pig

kidney

size,

taken

since

peptides

the

three

each

all of

above

sequence

into of

15-20 peptides

was

account

them

appeared

amino

acids

was

readily

for in in

determined

aligned

selecting the

within

elution

length. by

the

The automated the

primary

above

tryptic

region

of

complete Edman

strucdegrada-

structure

of

fructose-1,6-bisphosphatase.

Automated sequence

factor

Edman

degradation

of

the

tryptic

Lys-Leu-Asp-Val-Val-Ser-Asn-Glu-Val-Phe-Ser-Ser-Cys-Leu-Arg,

524

peptide

T-16

showed

the a

Vol. 133, No. 2, 1985

sequence

that

residues

72-86

was obvious

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

exhibits of pig

since

T-23

kidney

the

Lys-Leu-Asp-Val, cerevisiae

47% of amino

acid

identity

tryptic

peptide

a tetrapeptide

also

sequence

of residues

sequence

T-16

begins

present

of

The alignment with

in pig

the sequence

kidney

The analysis

(12)

and in 2.

of tryptic

peptide

Gly-Val-Tyr-Ala-Phe-Thr-Leu-Asp-Pro-Met-Tyr-Gly-

Glu-Phe-Val-Leu-Thr-Ser-Glu-Lys, sequence

the

fructose-1,6-bisphosphatase.

fructose-1,6-bisphosphatase.

gave the

with

which

180-199

of the

shows

pig

50% identity

kidney

with

enzyme.

the

The sequence

Leu-Leu-Tyr-G1u-Cys-Ala-Pro-Met-Ser-Phe-I1e-Val-G1u-G1n-Ala-Gly-Gly-Lys obtained

by automated

clearly

aligned

kidney

Edman degradation

this

peptide

with

including

the

and a comnon tripeptide

peptide

T-21

(Leu

through

positions

9 through

sequenced

CNBr fragment

Fig.

under

already

of the

established

The comparison

phatase.

of 410 amino

bisphosphatase

acid

subunit

degree

regulation.

2J.

of homology

gluconeogenic

catalyzing

most

sequence

likely

the

spinach

of the

determined

51% amino

P.B.

from

for

sequenced

is

peptides

78 out of an estimated

acid

an unusual

and F. Marcus, 525

spinach

fructose-1,6-bisphos-

chloroplast

different

the same precursor

Harrsch,

already

fructose-1,6-

sequence

of pig

identity

plus

similarity. origin

functions

kidney 15% of

This for

fructose-1,6-bisphosphatase,

this

of

since

obtained

a comnon evolutionary

but having

remarkably,

for

a 66% of overall

suggests

8 residues

information,

the

kidney

of the

and chloroplast

originated

of pig

residues

same reaction

Most

Rittenhouse,

sequence

giving

first

data

data

replacements,

are

Leu-Leu-Tyr-

of the

and aligns

reveals

of pig

fructose-1,6-bisphosphatase.

above

that

277-294

sequences

the

beginning

sequence

with

T-21

pentapeptide

of the

fructose-1,6-bisphosphatase conservative

the

Only

to the

of chloroplast all

of residues

new sequence

fructose-1,6-bisphosphatase

the

malian

with

provided

peptide

61% of the

Ala-Gly-Gly.

18 corresponded

2 summarizes

chloroplast

Indeed

Met)

tryptic

sequence

same beginning

Glu-Cys

high

the

fructose-1,6-bisphosphatase.

identical

total

of the

mamenzymes

and modes of

case of two enzymes but only unpublished

very

that

one of them (the results.

Vol. 133, No. 2, 1985 chloroplast however,

enzyme) that

be involved

contains

disulfide

a structural in the

1,6-bisphosphatase. involved

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

unique

region(s) regulatory

Work is

in the light-dependent

bonds. present

It only

properties

in progress activation

to locate

would in the

appear plant

of chloroplast the cysteine

of chloroplast

likely, enzyme may fructoseresidues

fructose-1,6-

bisphosphatase. ACKNOWLEDGMENTS We thank Dr. Judith Rittenhouse for her help in the immunological experiments, Dr. Roger Poorman, The Upjohn Company, Kalamazoo, Michigan, for providing us with an Apple IIe computer program for protein sequence comparisons, and Ms. Ida Edelstein for assistance with the enzyme purifiThis work was supported by grants from the U.S. Department of cation. Agriculture (83~CRCR-l-1299), and the National Institutes of Health (AM 26564). REFERENCES 1.

Benkovic, S.J. and DeMaine, MM. (1981) Adv. Enzymol. 53, 45-82. Tejwani, G.A. (1982) Adv. Enzymol. 54, 121-194. 32: Zimmerman, G., Kelly, G.J. and Latzko, E. (1978) J. Biol. Chem. 253, 5952-5956. 4. Weeden, N.F. (1981) J. Mol. Evol. 17, 133-139. 5. Halliwell, B. (1981) In: Chloroplast Metabolism, pp. 66-88, Oxford University Press, New York. 6. Buchanan, B.B. (1980) Ann. Rev. Plant Physiol. 31, 341-374. 7. Preiss, J., Biggs, M.L. and Greenberg, E. (1967) J. Biol. Chem. 242, 2292-2294. 8. Buchanan, B.B., Schurmann, P. and Kalberer, P.P. (1971) J. Biol. Chem. 246, 5952-5959. 9. Buchanan, B.B., Wolosiuk, R.A. and Schurmann, P. (1979) Trends Biochem. Sci. 4, 93-96. 10. Pradel, J., Soulie, J.-M., BUC, J., Meunier, J.-C. and Ricard, J. (1981) Eur. J. Biochem. 113, 507-511. 11. Marcus, F. Edelstein, I., Nishizawa, A.N. and Buchanan, 8.6. (1980) Biochem. Biophys. Res. Comnun. 97, 1304-1310. 12. Marcus, F., Edelstein, I., Reardon, I. and Heinrikson, R.L. (1982) Proc. Natl. Acad. Sci. USA 79, 7161-7165. Fisher, W.K., and Thompson, E.O.P. (1983) Aust. J. Biol. Sci. 36, 13. 235-250. 14. Zimmerman, G., Kelly, G.H. and Latzko, E. (1976) Eur. J. Biochem. 70, 361-367. 15. Nishizawa, A.N., Yee, B.C. and Buchanan, B.B. (1982) In: Methods in Chloroplast Molecular Biology (M. Edelman, R.B. Hallick, and N.-H. Chua, eds.) pp. 707-713, Elsevier Biomedical Press, Amsterdam. 16. Rittenhouse, J. and Marcus F. (1984) Anal. Biochem. 138, 442-448. Rittenhouse, J., Harrsch, 17. P.B. and Marcus, F. (1984) Biochem. Biophys. Res. Comnun. 120, 467-473. Rittenhouse, J., Chatterjee, T., Marcus, F., Reardon, I., and 18. Heinrikson, R.L. (1983) J. Biol. Chem. 258, 7648-7652. 19. Tarr, G.E. (1981) Anal. Biochem. 111, 27-32 . 20. Black, S.D. and Coon, M.J. (1982) Anal. Biochem 121, 281-285. 21. Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L. (1985) J. Chromatogr. 336, 93-104. 22. Engvall, E. (1980) Methods Enzymol. 70, 419-439. 526