oxygenase from Rhodospirillum rubrum

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 232, No. 1, July, pp. 280-295, 1984

Complete

FRED

Primary Structure of Ribulosebisphosphate Carboxylase/ Oxygenase from Rhodospirillum rubrum’*’ C. HARTMAN?

Biology Llivisicm,

CLAUDE

D. STRINGER,

AND

EVA H. LEE4

Oak RtXge National Laburatmq and the University of Tennessee-Oak Ridge School of Biomedical sciaces, Oak Ridge, Tenwee 37830

Graduate

Received

January

10, 1984, and in revised

form

March

19, 1984

Of the 14 cyanogen bromide fragments derived from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, four are too large to permit complete sequencing by direct means [F. C. Hartman, C. D. Stringer, J. Omnaas, M. I. Donnelly, and B. Fraij (1982) Arch. B&hem. Biophgs. 219,422-4371. These have now been digested with proteases, and the resultant peptides have been purified and sequenced, thereby providing the complete sequences of the original fragments. With the determination of these sequences,the total primary structure of the enzyme is provided. The polypeptide chain consists of 466 residues, 144 (31%) of which are identical to those at corresponding positions of the large subunit of spinach ribulosebisphosphate carboxylase/oxygenase. Despite the low overall homology, striking homology between the two species of enzyme is observed in those regions previously implicated at the catalytic and activator sites.

As the enzyme that initiates the competing pathways of photosynthetic COz reduction and photorespiration, ribulose-Pz6 carboxylase/oxygenase (EC 7.1.1.39) plays a central role in plant growth and productivity [see Refs. (1) and (2) for reviews].

As one facet of acquiring structural and mechanistic information about the enzyme, our laboratory has attempted to map its catalytic site with affinity labels (3-6). Given the enzyme’s structural complexities in addition to inherent difficulties in the interpretation of data derived from chemical modifications, we felt that it was important to determine whether residues implicated at the catalytic site were in fact located within conserved regions of primary structure. If compelling conclusions are to be drawn concerning invariance and hence essentiality of certain regions, highly diverse species that lack extensive homology should be examined. Ribulose-Pz carboxylases from most species are composed of eight large catalytic subunits (52,000 Da) and eight small subunits (14,000 Da) of ill-defined function (2). Although sequences of the large subunits of the carboxylases from maize (7), spinach (8), tobacco (9), Chlxzmgdumonas reinhardii (lo), Anabaena 7120 (ll), SynechococcusPCC6301(12), and Anaqstis ni-

’ The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. *Research sponsored jointly hy the Science and Education Administration of the U. S. Department of Agriculture under Grant 78-59-2472-O-1-161-1 from the Competitive Research Grants Office and by the Office of Health and Environmental Research, U. S. Department of Energy under Contract W-7405~eng26 with the Union Carbide Corp. s To whom all correspondence should be addressed. ’ Postdoctoral investigator supported by the U. S. Department of Agriculture grant through Subcontract 3322 from the Biology Division of the Oak Ridge National Laboratory to the University of Tennessee. 5 Abbreviations used: ribulose - Pp. D - ribulose 1,5-bisphosphate; Quadrol, N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine; PTH, phenylthiohydantoin. 0603-9861/84 Copyright All rights

$3.00

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

280

SEQUENCE

OF

R. rubrum

RIBULOSEBISPHOSPHATE

du.!&ns (13) (all of which contain eight large and eight small subunits) have been deduced from their corresponding gene sequences, little is gleaned about features essential to function because of >80% homology among these species. Thus, we have been attracted to ribulose-Pz carboxylase/ oxygenase from Rhxwbq%iUum rubrum for several reasons: (a) the organism (a purple, non-sulfur photosynthetic bacterium) is evolutionarily quite primitive (14), (b) the enzyme’s quaternary structure is unique among ribulose-Pz carboxylases consisting solely of two identical large subunits (15, 16), and (c) amino acid analyses suggested little homology between the R. rub-urn subunit and the plant-type large subunit (1’7). Our earlier publications on the enzyme’s primary structure provided the sequences of tryptic peptides accounting for all the cysteinyl residues (18) and the sequences of most of the fourteen cyanogen bromide fragments (19). Four of the latter, because of size, could not be sequenced completely by direct Edman degradation. Hence, in this paper we report cleavage of the four large fragments and subsequently the elucidation of their complete sequences. Coupled with the prior sequence information, this now provides the total primary structure of the R. r&rum carboxylase, the subunit of which is comprised of 466 residues and exhibits only 31% homology with the large subunit of the spinach enzyme. While our studies were in progress, the gene that encodes the R. rubrum carboxylase was cloned and sequenced (20). The independently determined primary structures are in excellent accord. EXPERIMENTAL

PROCEDURES

Materials Chemicals, buffers, and reagents required for amino acid analyses and for automated Edman degradations were purchased from either Beckman Instruments Inc. or Pierce Chemical Company. Additional materials and respective vendors were polybrene from Aldrich Chemical Company, ultrapure urea from Schwarz/Mann, sequenal-grade trifluoroacetic acid from Pierce Chemical Company, r’C]iodoacetic acid from New England Nuclear, carboxypeptidase A from Sigma Chemical Company, TPCK-treated trypsin and clostripain from Wor-

CARBOXYLASE

281

thington, and Staphylococcus auTeus VB protease from Miles Laboratories. The clostripain was stored at -80°C at 2 mg/ml in 0.02 M NH,HCOs/20% (v/v) glycerol (pH 8.0). Prior to use, the thawed solution was diluted with an equal volume of 0.02 M NH,HCOJ 0.02 M dithiothreitol (pH 8.0) and incubated for 1 h at 37°C in order to activate the protease (21). General methods. Ribulose-P, carboxylase/oxygenase from R. rubrum was purified to homogeneity and alkylated with [i’C]iodoacetic acid (sp act = 510 cpm/ nmol) to protect sulfhydryl groups by published procedures (16, 18). The carboxymethylated protein, which had a sp act = 2700 cpm/nmol corresponding to an incorporation of 5.3 mol of carboxymethyl groups per mol of subunit [each subunit contains five cysteinyl residues (18)], was cleaved with CNBr, and the four large fragments (C12, C14, Cl6, and C19) were isolated as described earlier (19). A Packard 3255 liquid scintillation spectrometer was used to measure radioactivity. Samples were diluted to 0.5 ml with water and then dissolved in 10 ml of ACS counting solution (Amersham Corporation). Urea was deionized by batchwise treatment with Bio-Rad AG 501-X8 resin. During column chromatography, peptides were detected by monitoring of the effluents at 215 nm with a Gilson Holochrome. In most cases, volatile buffers (e.g. NHIHCO-J were used during chromatography of peptides so that the purified peptides could be recovered in dry, salt-free state by lyophilization. With nonvolatile buffer systems (e.g. those containing urea), the purified peptides were first dialyzed against 1% (v/v) acetic acid by use of Spectrapor 2000 membrane tubing (Spectrum Medical Industries) and finally lyophilized. Proteol~ic digestions of CNBr fragwwnts Preceding the preparative digestions that are reported under Results, small-scale experiments were carried out to establish appropriate conditions. Progressions of the digestions were monitored by HPLC (Laboratory Data Control); fractions were collected and subjected to amino acid analyses so as to identify those peptides whose sequences were to be established. A 250 X 4.6mm column of Lichrosorb RP8 5 pm from Laboratory Data Control was used for HPLC of peptides; the solvent system consisted of 0.1% (v/v) aqueous trifluoroacetic acid (equilibration) and 0.1% (v/v) trifluoroacetic acid in acetonitrile (limit) as described by Mahoney and Hermodson (22). Amino acid ana&&. Total acid hydrolysis of peptides was achieved in evacuated (~50 pm Hg) sealed tubes with 6 N HCl/O.Ol M 2-mercaptoethanol at 110°C for 21 h. Hydrolysates were dried on a Speed Vat concentrator (Savant Instruments Inc.) and subjected to chromatography on a Beckman 121 M amino acid analyzer using Beckman’s “3-hour, single-column system.” The analyzer was hard wired to a PDP data acquisition system. Computer programs used in data collection and analysis were provided by S. S. Stevens

282

HARTMAN,

STRINGER,

and J. T. Holderman of the Oak Ridge National Laboratory. Sequence analyses. Purified peptides (IS-200 nmol) were subjected to automated Edman degradations with a Beckman 89OC sequencer, the vacuum system of which was modified according to Bhown et d (23). A liquid nitrogen cold trap was inserted between the low-vacuum pump and the vacuum manifold. Polybrene (2 mg) was added to the reaction cup and carried through one complete cycle, before the introduction of the peptide sample. This greatly reduces wash-out of the peptide during the repetitive degradations (24). The program used with the Quadrol buffer was that of Bhown et al (23) with several modifications. The buffer concentration was reduced from 0.5 to 0.1 M, the drying step after delivery of phenyl isothiocyanate was increased from 20 to 66 s, and the coupling steps and drying steps after delivery of benzene/ethyl acetate were from Beckman’s peptide program 030176. For the cleavage step, the reaction cup was placed at high speed during the addition of heptafluorobutyric acid up to the level of the undercut; the reaction cup was then brought to low speed for the duration of the cleavage step. Half of the fraction from each cycle was hydrolyzed in base for quantitation as free amino acid on the amino acid analyzer (25). Threonine and arginine appear as y-aminobutyric acid and ornithine, respectively, in base hydrolysates. Serine appears as a degradation product that coelutes with cysteic acid. Generally, the other half of each fraction was converted to the phenylthiohydantoin for identification by HPLC, this provided confirmation of the identifications made by amino acid analyses and distinguished aspartic acid and glutamic acid from the corresponding amides. A 250 X 4.6-mm Spherisorb ODS 5 pm column (Laboratory Data Control) with a solvent system comprised of a lo-50% (v/v) gradient of acetonitrile in 0.1% (v/v) aqueous phosphoric acid was used for HPLC. A portion of the PTH derivatives from those cycles tentatively identified as serine was subjected to acid hydrolysis, thereby converting serine to alanine (26). followed by amino acid analysis. For those peptides that were radioactive, 10% of each fraction from the sequencer was counted so as to identify carboxymethylcysteine; the remainder of the fraction was then processed as already described. RESULTS

Sequence of CNBr fragrmnt

C12. Direct

Edman degradation provided the sequence of the first 47 residues of fragment Cl2 (19). Subsequent to digestion of Cl2 with clostripain, a peptide (C12CL2) was isolated which represented segment 36-62 of Cl2 and hence overlapped with the known amino-terminal sequence. From the same

AND LEE

clostripain digest, the carboxyl-terminal peptide (ClZCLl, residues 63-70) was isolated. The carboxyl-terminal sequence -Lys-Ala-Hse-COOH was confirmed by digestion of intact Cl2 with carboxypeptidase (Fig. 1A). Salt-free, lyophilized peptide Cl2 (265 nmol) was dissolved in 2.65 ml of 0.02 M NH4HC03/2 M urea (pH 8.0), and to this solution at room temperature was added 53 pg of clostripain dissolved in 53 ~1 of dithiothreitol-containing buffer (see Experimental Procedures). After 2 h at room temperature, the digest was fractionated on DEAE-cellulose (Fig. 2A). The desired fragments Cl2CLl (150 nmol, 57%) and C12CL2 (150 nmol, 57%) appeared quite pure as judged by HPLC (Fig. 2B) and

1.0

0.5

It

I

2

4

8

4

5

I6

12 16 TIME (hr)

20

24

3

L

FIG. 1. Digestion of CNBr fragments Cl2 (A), Cl4 (B), and Cl9 (C) with carboxypeptidase A. In each case, the peptide was present at 20 nmol/ml in 0.05 M N-ethylmorpholinium hydrochloride12 M urea (pH 8.0) with carboxypeptidase at 0.2 mg/ml. Digestions of peptides Cl2 and Cl9 were carried out at 37’C, whereas digestion of peptide Cl4 took place at 24°C. Periodically, 75 ~1 of digest was diluted with 75 ~1 of ice-cold 0.2 M sodium citrate (pH 2.2), and the solution was then subjected to amino acid analysis.

SEQUENCE

OF

R.

rubrum

RIBULOSEBISPHOSPHATE

A

,/’1'

2s

VOLUME

283

CARBOXYLASE

/'

/’

Al3

I

i

r500’ 2

(ml1

02

/

E ‘” < o.oe 12CLl

004

P 5

,

30

5

30

TIME (min)

FIG. 2. (A) Ion-exchange chromatography of a clostripain digest of CNBr fragment Cl2 (265 nmol) on a column (1 X 25 cm) of Whatman DE-52 equilibrated with 0.01 M NHIHCOs (pH 8.0) and eluted with a 400-ml linear gradient of N)4HCOa (pH 8.0) as shown. (B) HPLC of the original digest (left), purified peptide Cl2CLl (center), and purified peptide C12CL2 (right) (see Experimental Procedures).

amino acid analyses (Table I in miniprint supplement). Data from Edman degradations of peptide Cl2CLl and of peptide C12CL2 (shown in Tables II and III, miniprint supplement) then provide the complete sequence of CNBr fragment Cl2 (Table I). Fragment Cl2 (70 residues) is considerably smaller than was estimated by amino acid analysis (110 residues) (19). This discrepancy is lessened if carboxymethylcysteine is assigned a value of 1.0 rather than basing the composition on homoserine as in the earlier report. In any event, the published molar ratios of amino acids agree closely with the presently established sequences.

Although an overlap between Cl2CLl and C12CL2 has not been obtained, amino acid analyses of the nondesignated peaks in Fig. 2A did not reveal additional unique fragments, and the gene sequence (20) showed that ClZCLl and C12CL2 are contiguous. Asn at position 24 was initially reported as Asp (19); this error was detected during the sequencing of an active-site peptide contained within Cl2 (6) and during the DNA sequencing (20). The assignment of Glu at position 46 instead of Gly as reported earlier (19) is established by having sequenced peptide C12CL2 and also agrees with DNA sequencing (20).

TABLE SEQUENCES

I

OF CNBr

FRAGMENTS”~

Peptide 10

Cl2

L;s-P&L&A

20

i a-G i y-T;r-Guy-T;r-V~l-A~=-~=-Aia-Aia-His-l-

IO YO C;s-T~=-T~r-A~p-A~p-~~-~=-A:g-Giy-V~l-A~p-Ai~-L~"-V~l-~~-Gi"-V~l-A~p-G ,* + + + 3.

*

*

+

+

60

Tyr-Pro-Val-Ala-Leu-Phe-Asp-Arg-Aan-Ile+ + + + * + + ~-------clZCLZ

+

+

50 i u-Aia-ArS-Glu-Leu-Thr-Lys-Ile-AlaT2$ * * * + + C

+

*

+

+

-+

70

+

+ * +----C1zcL1

+

+

*

I

30

*I)

50

Giy-A:S-P:o-G~~-V;;l-A~p-Giy-Giy-L~~-V~l-V~l-Giy-T~=-Ii~-Ii~-L~~-P~~-L~~-L~~-Giy-L~"-A~S-P~~-Ly~-P~~-Ph~-Al~+ + I+-----C14Tl 60 70 Glu-Ala-Cys-His-Ala-Phe-Trp-Leu-GLy-Gly-Gly-Asp-Phe-Ile-Ly~-Asn-A~p-Glu-P~o-Gln-Gly-Asn-Gln-Pro-Phe-Ala-P~o-Leu* + + + + -L + + + + -t + + + + + + + ----------C14Tl 1.14T2 btivator-site peptidI 90 Arg-Asp-Thr-Ile-Ala-Leu-V:l-A$+&p-A&l& -b + * .I. * .+ .+ + + + -14T2

+

+

+

-c

+

+

+

+

+

BO +

+

+

+

I

I 20

10

Cl6

*

P&-G i y-Pt;e-P;,z-Giu-A-&-Lk-Giy-A&,-A

i a-A&-V&-Iie-L&-T

t ~-A~~-Giy-G~y-Giy-A~~-Pik-Giy-H~~-Ii~-~p-Giy-P~O-

70

60

L;s-Giu-L~~-Aia-A:g-Aia-P~~-Glu-Ser-Ph~-P~~-Gly-A~p-Al~-A~p-Gl~-Il~-Ty~-P~~-Gly-T~p-A~S-Ly~-Al~-L~~-Gly-V~l+ + + + + + + + * + + + + + I-16~1-1

80

+

+

+

II-

I 90 Glu-Asp-Thr-ArS-Ser-ATa-Lk-P&-Ala

20

10

Cl9

A:S-A:S-Aia-cin-Aa'p-Gi~-~=-Giy-Giu-Ai~-L~~-L~~-P~~-S~=-~~-A~~-Ii~-T~=-Ai~-~p-A~p-P~~-P~~-Gi~-Ii~-Ii=-Ai*10 ho A:S-G~y-G~u-T;r-V~1-L~~-G~~-~~-P~~-Giy-Gi~-A~~-Ai~-S~~-Hi~-V~l-~~-L~~-L~~-V~l-~p-Gly-Ty~-V~l-~~-Gly-Al~,+ + + * + + + + -t +lgA; + C 70 60 Ala-Ala-Ile-Thr-Thr-Ala-Arg-Arg-Arg-Phe-P~~-A~p-A~~-~~-L~~-Hi~-Ty~-Hi~-ArS-Ala-Gly-Hi~-Gly-Ala-Val-Th~-Se~,+ -f + + -c .+ + + .+ + * ;gA1* + + * + -t + C 90 Pro-Gin-Ser-Lys-Arg-Gly-Tyr-Thr-Ala-Phe+ + + + + + + C19Al I

I

50 +

+

+

-t

+

*

+

+

80 +

+

*

+.

*

i.

*

+

T4b------1

a Arrows above the residues indicate sequence information derived from intact fragments by Edman degradation (-) or carboxypeptidase digestion (-). Arrows below the residues indicate sequence information derived from subfragments (described under Results) by Edman degradation (-). The “activator-site peptide” and peptides 11 and T4b were sequenced previously (18, 19, 27). b Hse denotes homoserine. 294

SEQUENCE

OF

R. rubrum

RIBULOSEBISPHOSPHATE

Sequence of CNBr fmgment Cl.4 The first 50 residues were established by direct Edman degradation of the intact fragment (19). The remainder of the sequence was provided by two peptides (C14Tl and C14T2) obtained from a partial tryptic digest. Trypsin (7 pg in 7 ~1 of H,O) was added to a solution of salt-free, lyophilized peptide Cl4 (120 nmol) in 700 ~1 of 0.02 M NH,HC03/2 M urea (pH 8.0) at room temperature. A second, equivalent portion of trypsin was added after 18 h, and the digestion was allowed to proceed for another 5 h, at which time the digest was chromatographed on DEAE-cellulose (Fig.

285

CARBOXYLASE

3A). Both peptides C14Tl (65 nmol, 54%) and C14T2 (59 nmol, 49%) appeared quite pure by HPLC (Fig. 3B); amino acid analyses suggested that the former, a radioactive peptide, was closely related to the cysteine-containing peptide T4c (18) and that the latter was derived from the carboxyl-terminus of Cl4 as it contained homoserine (Table I in miniprint supplement). Results of Edman degradations of peptides C14Tl and C14T2 are given in Tables IV and V (miniprint supplement). Confirmation of the carboxyl-terminal sequence of peptide C14T2 was provided by carboxypeptidase digestion of intact Cl4 (Fig. 1B). That peptides C14Tl and C14T2

0.6- A

I i ^ -500 rL

,+-0.15 /’

/’

,I’

8

-0.1

-IT s* -3oo<

-0.05 r.

-‘co

300 VOLUME

400

5al

k 1 t2 g

Y

i 200

c E

600

(ml) -1 00

Cl4T2

A

/

C14Tl

s 5 I!u ;,j/ 20

30

5

5

30

TIME (mid

FIG. 3. (A) Ion-exchange chromatography of a tryptic digest of CNBr fragment Cl4 (120 nmol) on a column (1 X 25 cm) of Whatman DE-52 equilibrated with 0.01 M NH,HCOa (pH 8.0) and eluted with a 400-ml linear gradient of NH,HCOs (pH 8.0) as shown. (B) HPLC of the original digest (left), purified peptide C14Tl (center), and purified peptide C14T2 (right) (see Experimental Procedures).

286

HARTMAN,

STRINGER,

are contiguous within Cl4 is proven by the prior characterization of a chymotryptic peptide which contains the site of carbamate formation (27) and which overlaps with peptides C14Tl and C14T2. The complete sequence of CNBr fragment Cl4 is given in Table I; the fragment is 92 residues long, in exact agreement with the number of residues based on amino acid analysis (19). Sequence of CNBr fragment Cl6. Automated Edman degradation provided the sequence of the first 61 residues in peptide Cl6 (Table VI in miniprint supplement). These results agree with those reported earlier (19) except for Glu (not Gln) at cycle 5 and Arg (not Gly) at cycle 35-corrections which confirm assignments deduced from DNA sequencing (20). Further successful degradation than observed before was achieved by improved repetitive yield with the Quadrol buffer system as compared to dimethylallylamine. The remainder of the sequence of Cl6 was obtained from a tryptic peptide (C16Tl) that overlapped the amino-terminal sequence provided by Edman degradation and peptide 11, the carboxyl-terminal fragment that results from cleavage of ribulose-P2 carboxylase with iodosobenzoic acid (19). Salt-free, lyophilized peptide Cl6 (200 nmol) was dissolved in 2.0 ml of 0.02 M NH4HC03/2 M urea (pH 8.0) and digested at room temperature with 20 pg of trypsin (dissolved in 20 ~1 of HzO); after 2 h, a second identical aliquot of trypsin was added to the reaction mixture, which was left for 4 additional hours. The digest was then fractionated by ion-exchange chromatography on DEAE-cellulose (Fig. 4A); HPLC profiles of the total digest and purified peptide C16Tl (70 nmol, 35%) are shown in Fig. 4B. Following amino acid analysis of peptide C16Tl (Table I in miniprint supplement), Edman degradation of this peptide (Table VII in the miniprint supplement) completed the sequence of CNBr fragment Cl6 (Table I). Fragment Cl6 is 90 residues in length; amino acid analysis indicated 88 (19). Sequence of CNBr fragment C19. Because of discrepancies between direct sequencing and deductions from DNA sequencing at

AND

LEE

positions 34,37, and 38 of peptide C19, Edman degradation of this CNBr fragment was repeated (Table VIII in miniprint supplement). In agreement with results of DNA sequencing (20), Glu (not Leu), Gly (not Phe), and Glu (not Gly) were identified at positions 34, 37, and 38, respectively. Digestion of peptide Cl9 with S. aureus V8 protease (28) yielded a 62-residue fragment (C19Al) which overlapped the sequence provided by Edman degradation of the intact peptide and a cysteine-containing peptide (T4b) reported earlier (19). Salt-free, lyophilized peptide Cl9 (500 nmol) was dissolved in 5.3 ml of 0.05 M ammonium acetate/2 mM EDTA/Z M urea (pH 4.0). To this solution were added, at 5-h intervals, five successive lSO-~1 aliquots of S. aureus V8 protease [l mg/ml in 0.05 M NH4HC03/2 mM EDTA/BO% (v/v) glycerol, pH 8.01. After a total reaction time of 30 h at room temperature, the digest was fractionated on carboxymethyl cellulose (Fig. 5A). Fractions from the center region of the major radioactive peak (C19Al) were pooled, dialyzed, and lyophilized. This peptide (180 nmol, 36%), which appeared highly pure by HPLC (Fig. 5B), was subjected to amino acid analysis (Table I in miniprint supplement) and Edman degradation (Table IX in miniprint supplement). Carboxypeptidase digestion of peptide Cl9 (Fig. 1C) provided the relationship between peptide T4b and carboxyl-terminal homoserine, thereby completing the sequence of CNBr fragment Cl9 (Table I). Fragment Cl9 is 96 residues in length, in good agreement with 98 as determined by amino acid analysis (19). DISCUSSION

Based on partial overlap information, on sequence data at the amino and carboxylterminal regions, and on limited sequence homology with the plant carboxylases, the 14 cyanogen bromide fragments derived from R. rubrum ribulose-Pz carboxylase/ oxygenase were aligned (19). The relative positioning of three short CNBr fragments were described as tentative, and two of these assignments (that of C2 and C6a)

SEQUENCE

R.

OF

r&ram

RIBULOSEBISPHOSPHATE

too

5

CARBOXYLASE

3oo 200 VOLUME

(ml)

30 5 TIME (mini

287

i 400

30

FIG. 4. (A) Ion-exchange chromatography of a tryptic digest of CNBr fragment Cl6 (206 nmol) on a column (1 X 25 cm) of Whatman DE-52 equilibrated with 0.01 M NH4HC03 (pH 8.0) and eluted with a 460-ml linear gradient of NI&HCOs (pH 8.0) as shown. (B) HPLC of the original digest (left) and purified peptide C16Tl (right) (see Experimental Procedures).

have since been shown to be incorrect (20). With these changes incorporated, completion of the sequencing of the four large fragments described herein (Table I) provides the total primary structure of the polypeptide chain, which is compared to that of the large subunit of the spinach enzyme (8) in Fig. 6. The sequence determined by direct methods agrees precisely with that deduced from the nucleotide sequence of the gene (20). Excepting the few

discrepancies noted and corrected under Results, the only other changes in the sequence shown in Fig. 6 compared to our partial sequence reported earlier are glutaminyl residues at positions 349 and 355 (within fragment C5) rather than glutamic acid. The polypeptide chain of the R. vub-urn enzyme (466 residues) is somewhat shorter than the large subunit of the spinach enzyme (475 residues) in that a 12residue insertion is observed at the NH2-

=: 1 I 1~.L+L.:J \-.-.-.-.-.-.I HARTMAN,

STRINGER,

A

AND

LEE

H

x400

C19Al

,

2 c

I

2z 200

.

,/I

f,,/.

id0

,

5

9

I

,

360

400

(ml)

,

I

30 TIME

z 1! t 0.1 -

,‘

260

VOLUME

FIG. 5. (A) on a column (pH 4.0) and HPLC of the

0.2

,/’

5

I

I

,

I

30

(min)

Ion-exchange chromatography of a S aureus digest of CNBr fragment Cl9 (500 nmol) (1 X 25 cm) of Whatman CM-52 equilibrated with 0.01 M ammonium acetate/6 M urea eluted with a 400-ml linear gradient of NaCl in equilibration buffer as shown. (B) original digest (left) and purified peptide C19Al (right).

terminus of the latter. Because of extreme sequence divergence, slight alterations in the illustrated alignments adjacent to short deletions and insertions can be envisioned (20). Of the 466 residues in R. rubrum carboxylase, only 144 (31%) are identical in the large subunit of the functionally analogous spinach enzyme. Thus, in addition to its unique quaternary structure among the many species of ribulose-Pz carboxylase examined to date, the R. r&rum enzyme ,also displays extraordinary sequence divergence in comparison to the maximal di-

vergence of 20% that has been observed among other species (7-13). With respect to the identification of residues or short segments of sequences that may be absolutely essential to structure, function, or regulation, attention can be focused on that relatively small percentage of the primary structure that is invariant. Studies with affinity labels have provided evidence for at least three regions of primary structure comprising the catalytic site. These are indicated.in Fig. 6 as “active-site peptide,” and in each case impressive homology is seen between the two

SEQUENCE

R.

OF

rubrum

RIBULOSEBISPHOSPHATE

289

CARBOXYLASE

16 -

Active-S;yo

Peptide

-

Cl4 210

Cl:

Cl411 I.

T4C

-Active-Sit

250

Peptide

Cl412

. cllv~tor-sole

PePlIds-

--XT

260

220

270

I80

nsn1 FY&k 0c4ag B ‘:D 0 [10 I” d” B !!I :)t% -14b-

6

370

Act,ve-Sata

YFTQSWVSTPGVLPVASQGIHVYH GYKACTPIISGQMNALR 360 cs 430

EA VOARNEGR &$RD4$~pV‘~”

Pcpttde

380

PALTEIFGDDSV PGFFENLGNANVILTAGGG

LQF

GGTLGHPW FGHID

I)

440

NAP AVAN PVAGARSLR

V

L

A

400

ClS

410

LAREGNTIIREATK YARE?3iELAR 00

AFE

S PELAAACEV “9J SFPGDADOIYP 0 ‘246o4fl

,

WRKALGVEDTRSALPA OE’KFn”AYDTV 4dO

Cl6 Cl611

c

-I

II

t

-

FIG. 6. Amino acid sequences of ribulose-Pz carboxylase/oxygenase from spinach (8) (upper) and R. rubrum (lower). Alignments were made by visual inspection. Residues identical in both enzymes are enclosed in boxes. Gaps, attributed to deletions or insertions, appear as blank spaces. Peptides C12CL2, CIZCLI, C14T1, C14T2, C19A1, and C16Tl were characterized in the present study. Other peptides whose notations begin with “C,” “ T,” or “I” were described previously (18, 19). Cysteinyl residues and residues identified at the active site or activator site by selective chemical labeling (discussed in text) are illustrated with larger type. Abbreviations: Asp, D; Asn, N; Glu, E; Gln, Q; Thr, T; Ser, S; Pro, P; Gly, G; Ala, A; Cys, C; Val, V; Met, M; Ile, I; Leu, L; Tyr, Y; Phe, F; His, H; Lys, K; Trp, W; Arg, R.

species. Lys-175 in the spinach enzyme has been selectively labeled by three different reagents-3 - bromo - 1,4 - dihydroxy -2 - butanone 1,4-bisphosphate (3), N-(bromoacetyl)ethanolamine phosphate (3), and pyridoxal S-phosphate (29, 30). The corresponding residue in the R. ruhum enzyme, Lys-166, is modified by pyridoxal phosphate with a high degree of specificity (31, 32). As is seen, an identical seven-residue sequence encompasses the reactive lysyl residue in the two species. A second activesite region contains Lys-334 in spinach

carboxylase and Met-330 in the h!. rubacm enzyme. The former is labeled by 3-bromo1,4-dihydroxy-2-butanone 1,4-bisphosphate (3) and the latter by 2-bromoacetylaminopentitol 1,5-bisphosphate (5). Although Met-330 is replaced by Leu-335 in the spinach enzyme and hence cannot function in catalysis, Lys-334 and eight other residues in its immediate vicinity are conserved. On the basis of species invariance, two different lysyl residues (Lys-175 and Lys-334) could participate in catalysis. Finally, a region encompassing His-44 in R. rubrum

HARTMAN,

STRINGER, TABLE

AND LEE

II

AMINO ACID SUBSTITUTIONSFOR CYSTEINYL RESIDUES BETWEEN SPINACH AND R. r&rum RIBULOSE-Pa CARBOX~LASE Residue Position (spinach enz)

Spinach

84

CYS

99 172 192 221 247 284 427 459

CYS CYS CYS CYS CYS CYS CYS CYS

Residue Position

R. rub-urn

Ala Ala Thr Phe Val Pro Ala Tw Ile

carboxylase is implicated by the selective labeling of this residue with 2-(4-bromoacetamido)anilino-2-dexoypentitol 1,5bisphosphate (6). Once again, the residue that is subject to selective alkylation by the affinity label cannot function in catalysis, because it is deleted in the enzyme from spinach. However, 11 of the 14 residues from positions 40 through 54 are conserved, strongly suggestive of functional participation of at least one of the invariant residues. Another region of high homology is seen at the activator site (labeled “activatorsite” peptide in Fig. 6). All ribulose-Pz carboxylases require COz and Me for activation; the process entails condensation of COz with a lysyl c-amino group (Lys-201 in the spinach enzyme) to form a carbamate that is stabilized by Me (33, 34). Chemically, the process is the same for the R. rubrum enzyme (27), and completion of this carboxylase’s primary structure identifies the COz acceptor as the lysyl residue at position 191. In each of the cases discussed, regions of primary structure, for which sound chemical evidence has suggested functional importance, are highly conserved in diverse species. Thus, credence is lended to universality of mechanism of ribulose-Pz carboxylases and their evolution from a single ancestral gene.

(R. rubrum

enz)

26 58 180 309 363

Rmbrum CYS CYS CYS CYS CYS

Spinach Ala Tw Val Ala Val

Given continuing speculation about the role of sulfhydryls in ribulose-Pe carboxylase (35, 36), it is of interest to note the complete lack of commonality in the location of cysteinyl residues in the two species (Table II). With the exception of the replacement of Cys-172 of the spinach enzyme with Thr in the R. rubrum, whereby each residue could participate in hydrogen bonding, all other substitutions for cysteines are with residues that cannot be functionally equivalent. Thus, it is difficult to visualize an essentiality of sulfhydryls unless in the plant-type enzymes they are indirectly involved in subunit/subunit interactions (the enzymes are devoid of disulfide bridges). ACKNOWLEDGMENT The authors thank Dr. Christopher Somerville of Michigan State University for communicating results prior to publication. REFERENCES 1. LORIMER, G. H. (1981) Annu. Rex Plant Physiol 32.349-383. 2. MIZIORKO, H. M., AND L~RIMER, G. H. (1983) Anna Rev. Bidem.

52, 507-535.

3. HARTMAN, F. C., NORTON, I. L., STRINGER, C. D., AND SCHLOSS, J. V. (1978) in Photosynthetic Carbon Assimilation (Siegelman, H. W., and Hind, G., eds.), pp. 245-269, Plenum, New York.

SEQUENCE

OF

R. r&rum

RIBULOSEBISPHOSPHATE

4. HARTMAN, F. C., FRAU, B., NORTON, I. L., AND STRINGER, C. D. (1981) in Proceedings of the 5th International Photosynthesis Congress (Akoyunoglou, G., ed.), vol. 4, pp. 17-29, Balaban, Philadelphia. 5. FRALJ, B., AND HARTMAN, F. C. (1983) Biochemistry 22, 1515-1520. 6. HERNDON, C. S., AND HARTMAN, F. C. (1984) J. BioL Chem. 259.3102-3110. ‘7. MCINTOSH, L., POVLSON, C., AND BOGORAD, L. (1980) Nature (London) 288, 556-560. 8. ZURAWSKI, G., PERROT, B., BOTTOMLEY, W., AND WHITFELD, P. R. (1981) Null Acids Res 9,32513270. 9. SHINOZAKI, K., AND SUGIURA, M. (1982) Gene 20, 91-102. 10. DRON, M., RAHIV, M., ROCHAIX, J. D. (1982) J. Mol. Biol. 162, ‘775-793. 11. CURTIS, S. E., AND HASELKORN, R. (1983) Proc Natl. Acad Sci. USA 80,1835-1839. 12. REICHELT, B. Y., AND DELANEY, S. F. (1983) DNA 2,121-129. 13. SHINOZAKI, K., YAMADA, C., TAKAHATA, N., AND SUGIURA, M. (1983) Proc. Natl. Acad Sci USA 80,4050-4054. 14. MCFADDEN, B. A., AND TABITA, F. R. (1974) Biosystems 6,93-112. 15. TABITA, F. R., AND MCFADDEN, B. A. (1974) J. Biol. Chem 249, 3459-3464. 16. SCHLOSS, J. V., PHARES, E. F., LONG, M. V., NORTON, I. L., STRINGER, C. D., AND HARTMAN, F. C. (1982) in Methods in Enzymology (Wood, W. A., ed.), Vol. 90, pp. 522-528, Academic Press, New York. 17. AKAZAWA, T., TAKABE, T., ASAMI, S., AND KoBAYASHI, H. (1978) in Photosynthetic Carbon Assimilation (Siegelman, H. W., and Hind, G., eds.), pp. 209-226, Plenum, New York. 18. STRINGER, C. D., NORTON, I. L., AND HARTMAN, F. C. (1981) Arch. B&hem. Biophys. 208,495501.

CARBOXYLASE

291

19. HARTMAN, F. C., STRINGER, C. D., OMNAAS, J., DONNELLY, M. I., AND FRAIJ, B. (1982) Arch, Biochem. Biophys 219,422-437. 20. NARGANG, F., MCINTOSH, L., AND SOMERVILLE, C. (1984) Mol. Gen Genet. 193,220-224. 21. MITCHELL, W. M., AND HARRINGTON, W. F. (1968) J. Biol Chem. 243,4633-4692. 22. MAHONEY, W. C., AND HERMODSON, M. A. (1980) J. BioL Chem. 255,11199-11203. 23. BHOWN, A. S., CORNELIUS, T. W., MOLE, J. E., LYNN, J. D., TIDWELL, W. A., AND BENNE’IT, J. W. (1980) Anal. Bioch,em. 102,35-38. 24. TARR, G. E., BEECHER, J. F., BELL, M., AND MCKEAN, D. J. (1978) Anal. B&hem. 84,622627. 25. SMITHIES, O., GIBSON, D., FANNING, E. M., GOODFLIESH, R. M., GILMAN, J. G., AND BALLANTYNE, D. L. (1971) Biochemistry 10, 4912-4921. 26. MENDEZ, E., AND LAI, C. Y. (1975) And Biochem. 68, 47-53. 27. DONNELLY, M. I., STRINGER, C. D., AND HARTMAN, F. C. (1983) Biochemistry 22, 4346-4352. 28. DRAPEAU, G. R., BOILY, Y., AND HOUMARD, J. (1972) J. Biol. Chem. 247, 6720-6726. 29. PAECH, C., AND TOLBERT, N. E. (1978)J. BioL Chem 253, 7864-7873. 30. SPELLMAN, M., TOLBERT, N. E., AND HARTMAN, F. C. (1979) in Abstract of Papers, Biol3,178th National Meeting of the American Chemical Society, Sept. 1979, Washington, D. C. 31. WHITMAN, W. B., AND TABITA, F. R. (1978b) Biw chemistry 17, 1288-1293. 32. HERNDON, C. S., NORTON, I. L., AND HARTMAN, F. C. (1982) Biochemistry 21, 1380-1385. 33. LORIMER, G. H., AND MIZIORKO, H. M. (1980) Bie chemistry 19, 5321-5328. 34. LORIMER, G. H. (1981) Biochemtitrg20,1236-1240. 35. WUDNER, G. F. (1981) Physiol Plant. (Cqwn~tm) 52,385-389. 36. BARNARD, G., ROY, H., AND MYER, Y. P. (1983) Biochemistry 22, 2697-2704.

Acid

Arg

to

set

1.0.

numbers

ZThe

0.1

in

denote

4.0

LYS

rrP

0.1

parentheses

(1)

1.G

0.1

HIS

1.1

2.6

0.1

TYr Phe

(4)

4.1

1.8

1.1

1.0

1.9

2.8

0.9

number

(2)

(1)

(1)

(2)

(3)

(1)

(4)

(1)

1.4

4.4

(1)

(3)

(1)

(3)

1.0

3.4

1.3

3.1

no. residu&

actual

ClZCL2

the

2.5

1.5

0.1

3.9

3.5

1.2

6.2

5.7

0.3

(1)

(1)

2.0

1.4

4.8

0.2

1.8

4.3

Leu

0.9

1.0

(1)

(1)

(2)

(1)

nmols found

Jle

Met

Val

GlY Ala

3-G

0.4

Clx

I.0

1.8

0.6

residues

no.

1.2

0.4

Ser

Cl2CLl

4.6

3.7

Pro

6.9

2.4

Thr

(Cd

nmols found

AW.

CYS

Homoserlne

Amino

Amino

of

I Purified

1.4

6.3

0.84

nmOlS found

1.0

0.9&

(1)

(1)

(2)

(1)

(3)

as determined

0.48

0.47

residues

1.6

0.93

1.6

2.7

2.6

(3)

0.91

(1)

1.1

2.0

(3)

(3)

(2)

2.7

3.1

3.5

1.9

1.1

0.1

3.0

3.4

1.9

Cl4T2

_

--

.,.

Peptides

by sequencing.

1.2

0.1

0.1

1.2

0.05

2.3

1.2

1.1

4.8

1.5

3.3

3.0

(1)

(1)

(1)

of

1.2 1.2

1.4

0.74

no. residues

Peptide

TABLE

0.1

Cl4Tl

Compositions

0.1

0.1

1.4

0.73

nmols found

Acid

1.0

1.0

1.9

1.0

1.1

4.2

1.3

2.0

3.2

1.2

5.3

0.70

no. residues

(1)

(1)

(2)

(1)

(1)

(4)

(1)

(3)

(3)

(1)

(5)

,.,-

(1)

^,

0.53

0.29

1.0

0.4&

0.05

0.45

1.1

1.2

1.0

1.0

0.58

0.05

0.91

mols found

-

C16Tl

-

1.1

0.59

2.0

1.0

0.92

2.2

2.4

2.0

2.0

1.2

1.9

no. residue8

_.,

(1)

(1)

(2)

(1)

(1)

(2)

(2)

(2)

(2)

(1)

(2)

,_,

3.9

1.7

3.0

3.1

2.4

2.3

O.&

3.6

8.1

4.7

1.4

1.9

2.4

3.8

3.1

-.

-

4.6

2.0

4.5

3.7

2.7 2.9

1.0

.I.

(5)

(2)

(5)

(4)

(3) (3)

(1)

(10) (5)

(6) 9.6 4.3

(2)

1.7 5.6

(3) (2)

(5) 2.9 2.3

(4)

3.7

(1) (1)

4.5

0.61 0.86

0.51 0.72

no. residue8

C19Al mols found

Ii m

i

kz "i

9

5

2

iz

EC

SEQUENCE

OF

R.

t-&rum

RIBULOSEBISPHOSPHATE

12.2 9.1 10.1 3.4 6.8 7.5 5.2 7.4 6.5 4.7

CARBOXYLASE

293

294

HARTMAN,

STRINGER,

AND

LEE

(n-1) 13.8

5.5

LO.0

2.1

12.8

2.3 c

L8.2 LO.4 14.8 11.5 5.3 7.6 8.L 6.7 5.3

145.8

32

127.L

33

14.7 -s

108.2

34

14.6

99.0

35

13.3

46.6

36

44.6

31

13.9

7.9

99.9

38

10.5

92.3

39

4.2

40.4

40

9.6

94.8

4,

8.4

22.9

42

9.3

80.8

43

3.2

67.4

44

5.6

59.3

45

8.0

46

4.L

63.5

47

6.B

53.8

48

5.1

49.4

49

I.8

46.9

50

3.0

53.8

51

4.2

42.0

52

4.6

47.3

53

1.5

6.9

54

0.8

58.9

55

0.7

14.7

56

0.8

27.4

57

2.8

15.6

58

1.9

25.9

59

I.9

24.9

60

0.5

21.0

6,

1.7

d

24.4

SEQUENCE

OF

R. rubrum

RIBULOSEBISPHOSPHATE

CARBOXYLASE

295