Plant Physiol. (1995) 109: 681-685

Pseudoreversion Substitution at Large-Subunit Residue 54 lnfluences the CO,/O, Specificity of Chloroplast Ribulose-Bisphosphate Carboxylase/Oxygenase'

Robert J. Spreitzer*, Craham Thow', and Cenhai Zhu3

Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664

Chlamydomonas reinbardtii mutant 31-4E lacks ribulose-l,5- bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) holoen- zyme due to a mutation in the chloroplast rbcL gene. lhis mutation causes a gly~ine~~-to-aspartate substitution within the N-terminal domain of the Rubisco large subunit. In the present study, photo- synthesis-competent revertants were selected to determine whether other amino acid substitutions might complement the primary de- fect. Revertants were found to arise from only true reversion or either of two forms of pseudoreversion affecting residue 54. One pseudorevertant has a gly~ine~~-to-alanine substitution that de- creases the accumulation of holoenzyme, but the purified Rubisco has near-normal kinetic properties. l h e other pseudorevertant has a gly~ine~~-to-valine substitution that causes an even greater de- crease in holoenzyme accumulation. Rubisco purified from this strain was found to have an 83% decrease in the V,,, of carboxy- lation and an 18% decrease in the COJO, specificity factor. These results indicate that small increases in the size of amino acid side chains can influence Rubisco assembly or stability. Even though such changes occur far from the active site, they also play a signif- icant role in determining Rubisco catalytic efficiency.

Rubisco (EC 4.1.1.39) exists within the chloroplasts of plants and green algae as a 560-kD holoenzyme made up of eight 55-kD large subunits (coded by the rbcL chloroplast gene) and eight 15-kD small subunits (coded by a family of RbcS nuclear genes) (reviewed by Spreitzer, 1993). Each large subunit has a C-terminal, a/p-barrel domain that interacts with the N-terminal domain of an adjacent large subunit to form the active site (Knight et al., 1990; Schreuder et al., 1993). CO, and O, are mutually compet- itive for RuBP within the active site, and the ratio of car- boxylation to oxygenation ultimately determines net pho- tosynthetic CO, fixation (Laing et al., 1974). Because the oxygenation reaction initiates photorespiration, a nones- sentia1 process wasteful of accumulated carbon (Somerville

' Supported by U.S. Department of Agriculture/National Re- search Initiative Competitive Grant No. 94-37306-0349 and pub- lished as paper No. 11140, Journal Series, Nebraska Agricultura1 Research Division.

Present address: Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland.

Present address: Department of Biochemistry, University of Arizona, Tucson, AZ 85721.

* Corresponding author; e-mail rjs8unlinfo.unl.edu; fax 1-402- 472-7842.

and Ogren, 1982), there is much interest in determining whether Rubisco might be engineered to increase carbox- ylation or decrease oxygenation.

The ratio of carboxylation to oxygenation is defined by the CO,/O, specificity factor (a = VcKo/VoKc) (Laing et al., 1974). This kinetic constant reflects the difference between the free energies of the carboxylation and oxygenation transition states (Chen and Spreitzer, 1991, 1992). Directed mutants of either prokaryotic Rubisco (expressed in Es- ckerickia coli [reviewed by Spreitzer, 19931) or alga1 Rubisco (recovered via chloroplast transformation [Zhu and Spre- itzer, 19941) have been used to assess the importance of individual large-subunit residues that coordinate with the carboxylation transition-state analog 2-carboxyarabinitol- 1,5-bisphosphate (Knight et al., 1990; Schreuder et al., 1993). Substitutions at the "active-site" residues eliminate or greatly reduce carboxylase activity, but severa1 have been found to influence R (Chène et al., 1992; Harpel and Hartman, 1992; Lee and McFadden, 1992; Lee et al., 1993a, 1993b; Larimer et al., 1994; More11 et al., 1994; Zhu and Spreitzer, 1994). These active-site-directed mutants have been particularly useful for understanding the catalytic mechanism of Rubisco (reviewed by Hartman and Harpel, 1994). However, because the active-site residues are essen- tia1 for maximal carboxylation, they may not be the most suitable targets for engineering a better Rubisco.

In contrast to higher plants and photosynthetic pro- karyotes, the green alga Chlamydomonas veinkavdtii has al- lowed the routine recovery of chloroplast rbcL mutations by random screening of photosynthesis-deficient mutants (reviewed by Spreitzer, 1993). Such mutants can be main- tained with acetate medium in darkness, a condition under which C. reinkardtii continues to synthesize a complete photosynthetic apparatus (Spreitzer and Mets, 1981). Anal- ysis of the rbcL mutants and their second-site suppressors has identified a number of regions and subtle interactions within the a/P-barrel C-terminal domain that influence i2 (Chen et al., 1988, 1991; Chen and Spreitzer, 1989; Thow et al., 1994). These regions may be better targets for attempt- ing to engineer an improved Rubisco.

A C. reinkardtii rbcL mutation named 314E was previ- ously identified that causes a Gly5*-to-Asp substitution within the large-subunit N-terminal domain (Thow and

Abbreviations: K,, K , for CO,; K,,, K, for O,; mt, mating-type locus; s2, C02/0 , specificity factor; RuBP, ribulose 1,5-bisphos- phate; V,, V,,, for carboxylation; V,, V,,, for oxygenation.

681

682 Spreitzer et al. Plant Physiol. Vol. 109, 1995

Spreitzer, 1992). Although this substitution eliminates theaccumulation of Rubisco holoenzyme, photosynthesis-competent revertants with partially defective Rubisco weresuccessfully selected and analyzed in the present study. Itappears that destabilization of the hydrophobia core of theN-terminal domain can also influence Rubisco ft.

MATERIALS AND METHODS

Strains and Culture Conditions

Chlamydomonas reinhardtii 2137 mt+ is the wild-typestrain maintained in our laboratory (Spreitzer and Mets,1981). The light-sensitive, acetate-requiring mutant 31-4Ewas recovered from mutagenized 2137 mt+ cells in a pre-vious study (Thow and Spreitzer, 1992). Mutant 31-4Elacks Rubisco holoenzyme due to a chloroplast rbcL muta-tion that causes a Gly54-to-Asp substitution within thelarge subunit (Thow and Spreitzer, 1992). All strains weremaintained on solid medium containing 10 mM sodiumacetate and 1.5% Bacto agar (Difco, Detroit, MI) at 25°C indarkness (Spreitzer and Mets, 1981). For biochemical anal-ysis, cells were grown in the same medium (without agar)on a rotary shaker at 220 rpm in darkness.

Revertant Selection

Independent, dark-grown clones of mutant 31-4E wereused in the reversion experiments to guard against theoccurrence of preexisting revertants. Spontaneous, photo-synthesis-competent revertants were selected by plating 2X 106 mutant cells per 100-mm Petri dish of minimalmedium at a light intensity of 80 /xmol photons irT2 s^1

(Chen and Spreitzer, 1989). In some reversion experiments,cells were mutagenized with methyl methanesulfonateprior to selection (Spreitzer and Chastain, 1987).

PCR, Gene Cloning, and DNA Sequencing

DNA isolation, PCR amplification of rbcL, cloning of thePCR products, and DNA sequencing were performed asdescribed previously (Thow et al., 1994).

Biochemical Analysis

About 2.5 x 109 cells were harvested by centrifugationand sonicated at 0°C for 3 min in 1 mM DTT, 10 mM MgCl2,10 mM NaHCO3, and 50 mM Bicine, pH 8.0. Protein contentwas determined by the method of Bradford (1976). Cellextracts were subjected to Sue-gradient fractionation(Spreitzer and Chastain, 1987) and SDS-PAGE (Laemmli,1970). The gels contained a 7.5 to 15% polyacrylamidegradient in the running gel and were stained withCoomassie blue after electrophoresis.

Values for fl were determined at 25°C by measuringcarboxylation and oxygenation simultaneously in 30-minreactions containing 10 /^g of Suc-gradient-purified en-zyme in 55 /J.M [l-3H]RuBP (8.6 Ci moP1), 2 mM NaH14CO3(5.0 Ci mor1), 10 mM MgCl2, and 50 mM Bicine (pH 8.3)(Jordan and Ogren, 1981a; Spreitzer et al., 1982). Because ofthe low activity of revertant R95-1C enzyme, 100 ;ug wasused in the reactions. Other kinetic constants for the puri-fied enzymes were determined as described previously(Chen et al., 1988; Chen and Spreitzer, 1989, 1991).

RESULTS

Genotypes of Photosynthesis-Competent Revertants

Mutant 31-4E is a light-sensitive, acetate-requiring strainthat lacks Rubisco holoenzyme due to a Gly (GGT) to Asp(GAT) substitution at large-subunit residue 54 (Thow andSpreitzer, 1992). In the present study, photosynthesis-com-petent revertants of mutant 31-4E were recovered on min-imal medium at a spontaneous frequency of about 4 per 109

cells. Following methyl methanesulfonate mutagenesis, therevertant recovery frequency increased to about 25 per 109

cells. Three spontaneous and 15 mutagen-induced rever-tants were saved for further analysis.

The rbcL gene was PCR amplified from each of therevertants, cloned, and sequenced through the originalmutant site. Eleven of the revertants were found to arisefrom true reversion, which would change Asp (GAT) backto Gly (GGT) at residue 54. Although these revertantstrains are of no further use for investigating Rubisco bio-chemistry, the observation of true reversion confirms the

AC G T (d) A C G T

—. ssar-

Figure 1. Reverse-complement DNA sequences of the chloroplast rbcL genes from wild type (a), mutant 31—4E (b), revertantR95-1C (c), and revertant R95-10A (d). The 31-4E rbcL gene has a G-C to A-T transition that changes large-subunit Gly54

(GGT) to Asp (GAT). The revertant R95-1C rbcL gene has an A-T to T-A transversion that changes the mutant Asp54 (GAT)to Val (GTT). The R95-10A rfacL gene has an A-T to C-G transversion that changes the mutant Asp54 (GAT) to Ala (GCT).

Pseudoreversion within the Rubisco N-Terminal Domain 683

1 2 3 4

LS-

SS-

Figure 2. SDS-PAGE of total soluble cell proteins from wild type(lane 1), mutant 31-4E (lane 2), revertant R95-1C (lane 3), andrevertant R95-10A (lane 4). Cells were grown with acetate mediumin darkness prior to extraction. Each lane received 60 ;ug of proteinextract. The gel was stained with Coomassie blue after electrophore-sis. LS, Large subunit; SS, small subunit.

correct assignment of the original 31-4E mutation (Thowand Spreitzer, 1992).

The remaining seven revertants were found to arise fromeither of two different mutations that also changed themiddle base of the codon for residue 54 (Fig. 1). Two ofthese "pseudorevertants" had a mutation that wouldchange Asp (GAT) to Val (GTT), and five had a mutationchanging Asp (GAT) to Ala (GCT). We use the term pseu-doreversion to describe these mutations because the orig-inal 31-4E mutation is eliminated but the wild-type DNAsequence is not restored. Although the rbcL sequence wasanalyzed for 18 revertant strains, no other mutation wasfound that would alter the codon for residue 54. Based onthe universal genetic code, this observation implies thatGlu (GAA, GAG), Asn (AAT), His (CAT), or Tyr (TAT)cannot substitute at residue 54 to restore Rubisco function.Furthermore, no intragenic suppressor mutation wasfound elsewhere within the rbcL gene, indicating that it isdifficult to structurally complement the Gly54-to-Asp sub-stitution by a change in some other residue of the primarystructure.

Biochemical Phenotypes of the Pseudorevertants

One strain from each of the two pseudorevertant classeswas saved for further study. Revertant R95-1C contains theGly54-to-Val substitution, and revertant R95-10A containsthe Gly54-to-Ala substitution. The entire rbcL gene wassequenced from each of these two revertant strains to con-firm that no other mutation was present.

Revertant R95-1C (Val54) grows substantially slowerthan wild type when compared on minimal medium in thelight, but revertant R95-10A (Ala54) is indistinguishablefrom wild type. All three strains grow at the same rate withacetate medium in darkness. When extracts of dark-growncells were fractionated on Sue gradients, both revertantswere found to have reduced levels of Rubisco holoenzyme.Revertant R95-1C (Val54) had about 20% and revertantR95-10A (Ala54) had about 50% of the wild-type level.Similar reductions in the levels of Rubisco subunits werealso observed when cell extracts were fractionated by SDS-PAGE (Fig. 2). Because unassembled Rubisco subunits arerapidly degraded within the C. reinhardtii chloroplast(Spreitzer et al., 1985; Thow et al., 1994), one assumes thatthe reduced level of subunits reflects the reduced level ofrevertant holoenzyme in vivo.

Kinetic constants were determined for wild-type andrevertant holoenzymes isolated from Sue gradients (TableI). Wild-type Rubisco had an (1 value of 61 ±3 but rever-tant R95-1C (Val54) Rubisco had an ft reduced to 50 ± 3.This 18% decrease in ft results primarily from an 83%decrease in Vc. Even though the K0/XC of the revertantRubisco was increased by 70%, indicating a decreased af-finity for O2 relative to CO2, this "improvement" was ne-gated by a 51% decrease in VC/V0. In contrast, Rubiscofrom revertant R95-10A (Ala54) had kinetic constants thatwere the same as those determined for wild-type Rubisco,except that Km (RuBP) was increased by about 2-fold(Table I).

DISCUSSION

Based on the x-ray crystal structure of spinach Rubisco(Knight et al., 1990), Gly54 resides within a-helix B and islikely to be in Van der Waals contact with the side-chainatoms of Cys84 and Cys" in /3-strands C and D, respec-tively. Interaction between these secondary-structure ele-ments contributes to the hydrophobic core of the large-subunit N-terminal domain. The loop region betweena-helix B and /3-strand C positions several residues intoclose contact with the carboxylation transition-state analog

Table I. Kinetic properties of Rubisco purified from wild type and revertants R95-1C and R95-10AStrains

Wild type (Gly54)R95-1C(Val54)R95-10A (Ala54)a The values are the

na

VCK,A61 ±50 ±58 ±

means ±

Kf" KO° K<n Ko/C

333

so (n -

V.uCO2

33 ± 433 ± 434 ± 5

1) of three

K/Vo1'

IJ.M O2 iimol h ' mg" ' protein381648369

± 20± 36± 19

enzyme preparations.

861580

± 8± 3± 5

11.519.610.9

5.32.65.3

Km"

,J.M RuBP11 ±8 ±

21 ±

233

h Calculated values.

684 Spreitzer et al. Plant Physiol. Vol. 109, 'I995

2-carboxyarabinitol-1,5-bisphosphate (Knight et al., 1990). These active-site residues include G1u6', Thr6', and Trp66.

C. reinhardtii mutant 3 1 4 E lacks Rubisco holoenzyme because a G l ~ ~ ~ - t o - A s p substitution introduces a charged, bulky side group that would destabilize the hydrophobic core of the large-subunit N-terminal domain (Fig. 1; Thow and Spreitzer, 1992). It would be difficult to complement both the charge and size of Asp54 by a compensatory substitution elsewhere in the large subunit, and only true or pseudorevertants were found via revertant selection in the present study. With regard to the pseudorevertants, either Ala or Val was found to substitute for the smaller Gly54 (Fig. 1). Whereas Ala decreased the accumulation of holoenzyme with little effect on catalysis, the even larger Val substantially decreased holoenzyme accumulation, lowered V,, and caused a reduction in R (Fig. 2; Table I).

It seems possible that the G l ~ ~ ~ - t o - V a l substitution in a-helix B affects Rubisco R by influencing the position of one or more active-site residues within the adjoining loop region. The significance of these residues (comparable to residues 60, 65, and 66 of the spinach large-subunit pri- mary structure) has been explored via directed mutagene- sis of Rubisco from the cyanobacterium Synechococcus (Morell et al., 1994) and bacterium Rhodospirillum rubrum (Smith et al., 1990; Lee et al., 1993a; Larimer et al., 1994). Nonconservative substitution at each residue has been found to decrease V , and R. However, the precise role of each active-site residue remains unknown because their replacement influences a number of kinetic constants and partia1 reactions (Lee et al., 1993a; Larimer et al., 1994; Morell et al., 1994).

In the present study, a Glys4-to-Val substitution within the hydrophobic core of the N-terminal domain has been found to decrease R (Table I). Perhaps other substitutions in this region might improve Rubisco catalytic efficiency. Natural variation exists within the secondary-structure el- ements (a-helix B and p-strands C and D) that contribute to the N-terminal core (Knight et al., 1990). For example, spinach Rubisco has Alas3, HisS6, and C Y S ~ ~ , but C. rein- hardtii Rubisco has C Y S ~ ~ , Asps6, and Ala99. Such variation in Rubisco core residues may account for the differences in catalytic efficiencies of Rubisco enzymes from different species (Jordan and Ogren, 1981b). Considering that the nature of residues far from the active site can influence catalysis (Table I; Chen et al., 1988), further examination of distant residues may reveal new strategies for engineering an improved Rubisco.

ACKNOWLEDCMENTS

We thank Carolyn M. OBrien and Seokjoo Hong for performing phenotypic analysis and assisting with the preparation of the figures.

Received May 24,1995; accepted July 6, 1995. Copyright Clearance Center: 0032-0889/95/ 109/0681/05.

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