oxygenase from Chromatium vinosum

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 254, No. 1, April, pp. 63-68,1987

The Nature of L8 and LISI Forms of Ribulose Bisphosphate Carboxylase/Oxygenase from Chromatium vinosum’ JOSE TORRES-RUIZ Biochemistry/Biophysics

Program,

Received September

AND

BRUCE A. MCFADDEN’

Washington State University,

Pullman,

Washington, 99164-4660

17,1986, and in revised form December 81986

Ls and L8Ss forms of ribulose bisphosphate carboxylase/oxygenase (RubisCO) have been prepared from Chromatium vinosum by the extremely mild method of centrifugal fractionation. Only the L& form is detectable in crude extracts of this organism. Both forms show immunological identity in double diffusion studies using antibody to L subunits of the L8SBform. L subunits from both L8 and La& enzymes are identical by the criteria of peptides observed after limited proteolysis and N-terminal sequence analysis. In addition, these subunits show regions of homology with L subunits from Rhodospirillum rubrum, Anacystis nidulans, and spinach. S subunits of the C. vinosum enzyme are completely homologous to those from A. nidulans and higher plants from the 18th through 25th residue, a stretch preceded in all cases by two basic amino acids. 0 1987 Academic

Press, Inc.

In most autotrophs the enzyme D-ribulose l,&bisphosphate carboxylase/oxygenase (RuBisCO? EC 4.1.1.39) catalyzes primary COz fixation. The dominant form of this enzyme is composed of eight large (L) 55,000-Da subunits and eight small (S) l&000-Da subunits (1). More than a decade ago, however, RuBisCO was isolated from Rhodospirillum r&rum in an Lz form (2). In recent years it has become evident that genes for the L subunits of Lz and L&$ forms probably arose from a common ancestor (1, 3) as first postulated in 1973 (4). Presumably S subunits were added later in evolution but prior to the integration of the S subunit gene into the nucleus of most eukaryotic autotrophs (1).

Two forms of ribulose bisphosphate carboxylase were first isolated from a single organism, Rhodopseudomonas sphaeroides, by Gibson and Tabita. Form I was an LsSstype enzyme and form II, of uncertain aggregation state, lacked S subunits (5). Recently the form II gene has been used as a heterologous probe to detect colonies of Escherichia coli carrying a recombinant plasmid containing genes for form I (6). We have isolated highly active La and L& enzymes from Chromatium vinosum and postulated that the latter could be converted to the Ls enzyme during purification (7). We now present evidence that this hypothesis is correct in accounting for dual forms of RuBisCO from C. vinosum. MATERIALS

i This research was supported in part by Grant GM19972 and by a Minority Access to Research Careers Fellowship (to J.T.) from the National Institute of Health. ‘To whom correspondence should be addressed. 3 Abbreviations used: RuBisCO, D-ribulose 1,5-biphosphate carboxylase/oxygenase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol.

AND

METHODS

Bacterial strain and growth conditions. C. vinosum (strain D) obtained from M. Madigan of Southern Illinois University in Carbondale was grown using the HCOa-/SaO;a/Na$ medium of Hurlbert and Lascelles (8). Precultures of 500 ml were used to inoculate 13-1 carboys containing the same medium and allowed to grow for 5 days at 30°C with illumination before harvesting by continuous centrifugation. Cells were 63

0003-9861/87 $3.00 Copyright All rights

Q 1987 by Academic Press. Inc. of reproduction in any form reserved.

64

TORRES-RUIZ

AND

washed once in MEMMB buffer (50 mM MOPS, 0.1 mM EDTA, 1 m&f MgCla, 1 mM B-mercaptoethanol, and 50 mM NaHCOs, adjusted to pH 7.3 at W”C), and collected by centrifugation at 4300~. The washed cells were then weighed and stored at -20°C. Enzyme isolation and subunit dissociation Isolation of the Ls and L&, forms of RuBisCO was achieved from single cultures of C vinosum as described in detail previously (7) and as summarized in Fig. 1. Purification of the L subunits from the L& form was accomplished by dissociation of the native enzyme in 50 mM sodium phosphate (pH 7.5) containing 0.9% NaCl, 0.5% NaDodSO, (SDS), and 5 mM /3-mercaptoethanol for 2 h at 20°C, followed by chromatography on a Sephadex G-100 column (7). The protein fractions containing L subunits were pooled and excess SDS was removed by the method of Vinagradov and Kapp (9) using the ion retardation resin, AGllAS, from Bio-Rad. Electwxlution and amino acid sequencing analysis. The S subunits as well as the L subunits derived from either the Ls or L&$ enzymatic forms of RuBisCO from C. winosum were subjected to partial amino acid se-

MC FADDEN

quence analysis. L and S subunits were obtained by electroelution from a SDS-polyacrylamide gel polymerized from 12.5% acrylamide by standard methods after electrophoresis of 350 pg of L& enzyme (10). After electrophoretic elution from the gel matrix, the protein solutions were lyophilized, dissolved in 50 pl doubly distilled water (ddHsO), and after addition of 400 ~1 ice-cold ethanol, protein precipitation was allowed to proceed at -20°C for 8 h. The polypeptides were collected by centrifugation for 5 min at 12,000 rpm using a Beckman microfuge and redissolved in minimal volumes (30-70 ~1) of sterile ddHz0. Polypeptide samples (0.5-l nmol) were subjected to Nterminal amino acid sequence analysis using an Applied Biosystems gas phase sequenator.

Nondenaturing polyacrylamide gel electrophoresis and immunoblotting. Polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions was performed in a gel slab polymerized from 6% Bio-Rad ultrapure-grade acrylamide. Protein samples (25-50 pg) were subjected to electrophoresis for 5 h at 20 mA. The unstained gel was placed adjacent to a BioRad sheet of nitrocellulose paper and electroblotted

30 GRAMS OF WET-PACKED A

SONIC

c.

Y1M.%M

CELLS

TREATtlENT/CENTRIFUGATION

AT 17,000

G

SUPERNATANT MADE 10X GLVCOL

B

IN

POLYETHYLENE

(PEG)-6000

CENTRIFUGED 1 MR.

'_i PELLET

AND

AT 35,000

G FOR

l

SUPERNATANT

,

c 1 ""'"';fik;;NT

PELLET D

DISSOLVED

IN

BUFFER

E

0.2k0.8M

F

CHROHATOGRAPHV

FRACTIONATION

L&

BY VERTICAL

SUCROSE

GRADIENT

SEDIMENTATION AT 290,000

ON DEAE-SEPHADEX

INTO

A LINEAR

G FOR 130

NIN

AT '4°C

A-50

I ENZYME

*ALTERNATIVELY, 175,000

WHEN SEDINENTATION

G (INSTEAD

SUPERNATANT

0~ 35,000

ARE CARRIED

IN

STEP B Is

CONDUCTED

G) AND THE OTHER OPERATIONS

OUT EXACTLY

AS DESCRIBED>

AT ON THE

THE L8 ENZYNE

RESULTS.

FIG. 1. Flow scheme illustrating the isolation (Torres-Ruiz and McFadden, 1985).

of L&

and Lg forms of RuBisCO from C tinoaum

RUBISCO

FROM

for 12 h at 30 V in a Tris-glycine-methanol transfer buffer (11) containing 0.1% SDS. After transfer (12), rabbit antibodies to the L& form of RuBisCO from C. vinosum (1:1500 dilution) and biotinylated goat antibodies against rabbit IgG (1:X0 dilution) were incubated with the nitrocellulose sheet. Specific binding of antibodies to RuBisCO was detected by the avidin-biotinylated horse radish peroxidase system (Vector Laboratories bulletin, “Immunodetection of Antigens on Nitrocellulose Using the Vectastain ABC Kit”). Analysis of limited pro&o&s. Peptide analysis after limited proteolysis was achieved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis essentially as described by Cleveland et al. (13). Purified L subunits (50 pg) derived from either the Lg or the La& form in 10 mM Tris-HCl, pH 7.0 (25”C), containing 0.1% SDS and &mercaptoethanol, were loaded into a stacking gel above an SDS-polyacrylamide slab polymerized from 15% acrylamide. A protein solution in a given sample well was overlaid with 2.5 ag of a given protease: trypsin (tolylsulfonyl phenylalanyl chloromethyl ketone-treated), chymotrypsin (L-5amino-1-(ptoluenesulfonyl) amidopentylchloromethy1 ketone-treated), or endoproteinase Glu-C (protease VS) from StaphyZocoecus aurews V8. Chymotrypsin and trypsin were obtained from Sigma Co., and protease V8 was from Boehringer Mannheim Biochemicals. Electrophoresis was started at 20 mA and allowed to continue until the tracking dye was approximately 1 cm above the interface of the stacking and separation gel. At that point, electrophoresis was stopped to allow proteolysis for 20 min in the case when chymotrypsin or protease V8 had been used or for 30 min in the case of trypsin. Electrophoresis was then resumed until the tracking dye reached the bottom of the gel. The gel was stained in a mixture of water:methanol:acetic acid (5:5:1, v/v/v) containing 0.1% Coomassie brilliant blue overnight, and then destained several times with the mixture of water: methanol:acetic acid. RESULTS

The fm of RuBisCO in crude extract. A Western blot of soluble proteins in a crude extract of C. vinosum subjected to electrophoresis in a nondenaturing polyacrylamide gel slab is shown in Fig. 2. Antibodies to La& RuBisCO from C. vinosum detected only the L&3* form of the enzyme in

1234567

8

9

Chromatium

65 1

23

4

56

669, 440, 232, 140 )

67,

FIG. 2. PAGE analysis of RuBisCO and a crude cellfree preparation of C. tinosum in a gel slab polymerized from 6% acrylamide. Samples l-4 were stained with Coomassie brilliant blue after PAGE. Lane 1 represents standards (with molecular mass shown in Kda) from top to bottom: thyroglobulin, ferritin, catalase, lactate dehydrogenase, and bovine serum albumin. Lanes 2, 3, and 4 represent, respectively, Ls RuBisCO, L& RuBisCO, and the 1’7,000~ supernatant from C. tinosum cells. Lanes 5 and 6 represent Western blots of lanes 3 and 4, respectively, using antibodies to the LsSr form of RuBisCO.

the 17,OOOgsupernatant from cell-free preparations (Fig. 2), although in separate experiments these antibodies cross-reacted with the Ls form. Immunod@?k3n analysis of L subunits. Immunodiffusion studies of the Ls and L&S8 enzymes using antibodies to L subunits isolated from the L8Sa form (‘7) revealed that these enzyme forms were immunologically indistinguishable. In immunodiffusion of the LB and LsSs enzymes against antibodies to the latter, a strong precipitin band was observed for each pair with a spur at the junction. N-terminal sequence analysis of L sub units. Results of the sequence analysis of L subunits from the Ls form (top line) and L&$ form (second line) of RuBisCO from C. vinosum are shown below:

10 11 12

13 14 15 16

Ser-Lys-Thr-Tyr-Ser-Ala-Gly-Val-Lys-Glu-Tyr-Arg-Glu-Thr-~r-~r-Met-Pro-Asn-TyrSer-Lys-Thr-Tyr-Ser-Ala-Gly-Val-Lys-Glu-~r-Arg-Glu-Thr-~r-~r

PerI

lGlu1

1'7 18

19 20

66

TORRES-RUIZ

AND

Although secondary products (shown in brackets) were inexplicably observed in the 12th and 14th cleavages cycles for L subunits derived from the LsSs form, the primary cleavage products were identical in the lgresidue N-terminal sequence of L subunits from both Ls and LsSs enzymes. Proteolytic analysis of Lsubunits. Pep-

MC FADDEN

tide profiles shown in Fig. 3 were identical for L subunits from either Ls or LsSsforms after treatment with trypsin, chymotrypsin, or protease V8 from S. aureus. N-terminal analysis of S subunits. The amino terminal sequence of the S subunit from the LsSsform of RuBisCO from C! wirwsum was also determined and is shown below:

Ser-Glu-Met-Glu-Asp-Tyr-Ser-Ser-Thr-LeuGlu -Asp-Val-Asn-Ser-Arg1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Lys-Phe-Glu-Thr-Phe-Ser-Tyr-Leu-Pro-Ala-Met-Asp-Trp-ArgIle -Arg17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Lvs-Gln-Val-Glu-Tvr-Phe-Val-Ser-Lys-Gly--Asn-Pro -Ala- Be -Glu 33 34 35 36 3;7 38 39 40 41 42 43 44 45 46 47 48

DISCUSSION

In our prior research, it was observed that a polyethylene glycol (PEG)-6000treated extract of C. uinosum yielded only the L&S8form of RuBisCO after centrifugation at 35,000g and subsequent purification. Alternatively, centrifugation of this fraction at 175,OOOg followed by the same purification steps yielded only the Ls form.

a

b

FIG. 3. Peptides arising from units derived from L& (left in in each pair) after SDS-PAGE. and (d), trypsin, chymotrypsin, used, respectively. Display (a) after SDS-PAGE.

c

d

proteolysis of L subeach pair) or L8 (right In displays (b), (c), and V8 protease were shows each L subunit

This led to the following hypothesized dissociation of RuBisCO (7): L&&m = LB+ Ssm where Ssm represents Seunits that are associated with small membrane-derived vesicles. It was presumed that Ssm vesicles are quantitatively removed by centrifugation for 1 h at 175,000g in the viscous medium containing PEG-6000. The recovery of the LsSs species free of the Ls form after purification of the 35,000g supernatant implied that the L&S8form predominated in the original cell-free preparation and that it was in equilibrium with the Ls form as shown. The present research establishes that indeed only the L&S8form of RuBisCO is detectable in crude extracts of C vinosum. Moreover, the N-terminal sequences and proteolytic products of L subunits are identical whether they are derived from Ls of L&S, species, suggesting that there is one structural gene for L subunits in this organism and that the Ls enzyme is indeed derived from the L,& form. The present limited sequencing results suggest that the L subunit of Chromatium RuBisCO is similar to deduced sequences of the N-terminus of counterparts from the cyanobacterium Anacgstis nidulans 6301 (14) and spinach (Zurawski et aC, 1981) as shown below:

RUBISCO

FROM

6’7

Chrcnnatium

1 11 16 RhodospirillumMDQSSRY6VNLALKEEDL rubrum 1 6 11 16 21 spinach MSPQTQTKASVEFKAGVKDYKLTYY~PEY 1 6 11 16 21 A. nidulans M*PKTQSAA. .GYKAGVKDYKLTYYTPDY 1 6 11 C vinosum .S.KTYS... l . . .AGVKEYRETYYMPNY In the information shown, dots correspond to deleted residues when aligned sequences are compared to that deduced for spinach L subunits. Curiously the L subunit of the Chromutium enzyme is more like that from spinach or A. nidulans that that from the anoxygenic photosynthetic bacterium, Rhodospirillum rubrum (15). It has been suggested that translation of spinach large subunits is initiated at methionine but that a post-translational processing event cleaves the protein between Lys-14 and Ala-15 (16) on the basis of in vitro translation studies (17). Following this lead, Shinozaki et al. (14) have postulated an

Spinach

A. nidulans

6

MQVWP

PLGLK

MSMKT

2 C.winosum

1

SEMQDY

7 SSTLE

analogous processing site in RuBisCO from 6301 between Lys-11 and Ala12. Our data establish that there is no analogous processing site in the aligned region from C. vinosum L subunits although there are limited sequence similarities upstream from Ala-6. These observations raise a question about the physiological significance of the postulated processing. In connection with S subunits, the sequence of this polypeptide from RuBisCO of C. vinosum is compared below with that from spinach (18) and that deduced for the enzyme from A. nidulans (19):

11

16 21 26 __-_-------K; FETF SYLPP L ; TTEQ LLAEV , 1

11 ;

LPKER

R; FETF SYLPP L I I I 17 I 22 27

DVNSR

16

21 I

26

j SDRQ IAAQI I I I I 32

Kj_F_E_TF_S_Y_L_P_A_MiDWRI RKQVE

Shinozaki and Seguira (19) have stressed that stretches Phe-12 through Leu-21 and Tyr-54 through Phe-63 are completely conserved in S subunits from A. nidulans and higher plants. These workers have suggested that those regions may play an important role in binding S to L subunits and/ or in catalytic function. The first conserved sequence is boxed for comparison with that of C. vinosum S subunits. In the latter, AlaMet is substituted at positions corresponding to 20 and 21 for Pro-Leu in a sequence preceded by Pro. It seems clear that the

16

A. niduluns

6

12

26

31 NYLLV

31 EYMIE

37 YFVSK

36 KGWIP

36 QGFHP

42 G-NPA

41 PL

41 LI

47 IE

conserved boxed region probably does not contribute to catalytic activity of RuBisCO because the L8 form of this enzyme form C. vinosum has essentially the same activity as the L8Ssform (7). Another noteworthy feature of the N-terminal region of S subunits is the pair of basic residues which precedes the conserved sequence shown in the box. This is also seen in all higher plant S subunits. Finally four basic residues are seen in the 21-residue stretch after the conserved region in C. vinosum S subunits

68

TORRES-RUIZ

AND

(in comparison with one in the spinach and A. nidulans regions), suggesting that these residues may provide additional sites of cleavage in comparative studies employing limited tryptic hydrolysis. Recently the genes for L and S subunits of RuBisCO from C. vinosum have been cloned and expressed in Eschemkhia coli (20). An eludication of the deoxynucleotide sequence is in progress (H. Kobayashi, personal communication). Moreover, studies of crystals which diffract to at least 3.0A resolution have been initiated with the C. vinosum enzyme (21). We anticipate information complementary to ours that will enhance our understanding of the quaternary structure and S subunit function of RuBisCO from this organism. ACKNOWLEDGMENTS Financial support from Grant GM-19,972 and from a Minority Access to Research Careers Fellowship from the National Institute of Health (to J.T.) is gratefully acknowledged. We also thank Dr. Paul Bishop of our Biochemistry/Biophysics Program for the peptide sequence analyses. REFERENCES 1. MCFADDEN, B. A., TORRES-RUIZ, J., DANIELL, H., AND SAROJINI, G. (1986) Phil Trams R. Sot B 313,347-358. 2. TABITA, F. R., AND MCFADDEN, B. A. (1974) J. Bid Chem. 249,3459-3464. 3. MCFADDEN, B. A., AND MAJUMDAR, P. K. (1984) in Microbial Growth on Ci Compounds (Crawford, R. L., and Hanson, R. S., Eds.), pp. 14-20, Amer. Sot. Microbial., Washington, DC.

MC FADDEN

4. MCFADDEN, B. A. (1973) Badrid Rev. 37, 289319. 5. GIBSON, J. L., AND TABITA, F. R. (1977) .I. BioL chmh 252,943-949. 6. GIBSON, J. L., AND TABITA, F. R. (1986) Gene 44, 271-278. 7. TORRES-RUIZ, J., AND MCFADDEN, B. A. (1985) Arch. MicrobtbL 142,55-60. 8. HURLBERT, R. E., AND LASCELLES, J. (1963) J. Gm MicrobioL 33,445-458. 9. VINOGRADOV, S. N., AND KAPP, 0. H. (1978) And Biochem. 91.230-235. 10. HUNKAPILLER, M. W., LUJAN, E., OSTRANDER, F., AND HOOD, L. E. (1983) in Methods in Enzymology 91. pp. 2237-236, Academic Press, New York. 11. TOWBIN, H., STAEHELIN, T., AND GORDON, J. (1979) Proc. NatL Acad Sci USA 76,4350-4354. 12. JOHNSON, D. A., GAUTSCH, J. W., SPORTSMAN, J. R., AND ELDER, J. H. (1984) Gem And Tech% 12, 3-8. 13. CLEVELAND, D. W., FISCHER, S. ‘G., KIRSCHNER, M. W., AND LAEYMLI, U. K. (1977) .I Bid C%em 252,1102-1106. 14. SHINOZAKI, K., YAMADA, C., TAKAHATA, N., AND SUGIURA, M. (1983) Proc NatL Acad Sci USA 80,4050-4054. 15. HARTMAN, F. C., STRINGER, C. D., AND LEE, E. H. (1984) Arch, B&hem, Biophys. 232,280-295. 16. ZURAWSKI, G., PERROT, B., BOTTOMLEY, W., AND WHITFIELD, P. R. (1981) Nucleic Acids Res. 9, 3251-3270. 17. LANGRIDGE, P. (1981) FEBS Leti 123,85-89. 18. MARTIN, P. G. (1979) Au&. J. Plant PhgsioL 6,401408. 19. SHINOZAKI, K., AND SUGIURA, M. (1983) Nucleic Acids Res. 11,6957-6964. 20. VI&E, A. M., KOBAYASHI, H., TAKABE, T., AND AKAZAWA, T. (1985) FEBS I&t. 192.283288. 21. NAKAGAWA, H., SUGIMOTO, M., KAI, Y., HARADA, S., MIKI, K., KASAI, N., SAEKI, K.. KAKIJNO, T., ANLI HORIO, T. (1986) J. MoL BioL 191,577-578.