Characterization of the heterooligomeric red-type activase from red algae

Nitin Loganathana, Yi-Chin Candace Tsaia, and Oliver Mueller-Cajara,1

aSchool of Biological Sciences, Nanyang Technological University, Singapore 637551

Edited by Sue Wickner, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved October 28, 2016 (received for review July 2, 2016)

The photosynthetic CO2-fixing ribulose 1,5-bisphosphate car- Three distantly related classes of Rcas (green, red, and CbbQO boxylase/oxygenase (rubisco) is inhibited by nonproductive binding types) have been identified so far (13–16). They all belong to the + of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar superfamily of AAA (ATPases associated with various cellular phosphates. Reactivation requires ATP-hydrolysis–powered remodel- activities) (17) and function as ring-shaped hexamers that ing of the inhibited complexes by diverse molecular chaperones couple the energy of ATP hydrolysis to rubisco remodeling. known as rubisco activases (Rcas). Eukaryotic phytoplankton of the CbbQO requires one adaptor CbbO to associate with the red plastid lineage contain so-called red-type rubiscos, some of which AAA hexamer CbbQ6 to function (15). Common themes in the have been shown to possess superior kinetic properties to green-type activation mechanism are emerging (such as manipulation of rubiscos found in higher plants. These organisms are known to en- the large subunit C terminus for red-type Rca and CbbQO), al- code multiple homologs of CbbX, the α-proteobacterial red-type acti- though clear differences are also apparent (3, 12, 15). vase. Here we show that the gene products of two cbbX genes Following the primary endosymbiotic event, the green plastid encoded by the nuclear and plastid genomes of the red algae lineage toward green algae and plants retained the green-type form Cyanidioschyzon merolae are nonfunctional in isolation, but together IB rubisco from the cyanobacterial ancestor. In contrast, the chlo- form a thermostable heterooligomeric Rca that can use both α-pro- roplast genome of the red plastid lineage acquired a red-type form I teobacterial and red algal-inhibited rubisco complexes as a substrate. rubisco operon including the red-type Rca-encoding cbbX gene The mechanism of rubisco activation appears conserved between the from proteobacteria, probably via horizontal gene transfer (18, 19). bacterial and the algal systems and involves threading of the rubisco All red-lineage phytoplankton for which data are available appear large subunit C terminus. Whereas binding of the allosteric regulator to encode an additional cbbX gene in the nucleus (20). RuBP induces oligomeric transitions to the bacterial activase, it merely Inhibition data on form ID rubiscos from red lineage eukaryotic enhances the kinetics of ATP hydrolysis in the algal enzyme. Muta- phytoplankton is limited. Rubisco from a number of species formed tional analysis of nuclear and plastid isoforms demonstrates strong inhibited complexes of varying stability with RuBP (21), but in more coordination between the subunits and implicates the nuclear- detailed work, the enzyme from the red algae Galdieria sulphuraria encoded subunit as being functionally dominant. The plastid-encoded was reported to exhibit high inhibition constants (22). Low rubisco subunit may be catalytically inert. Efforts to enhance crop photosyn-

activation states in rapidly extracted soluble lysates from various BIOCHEMISTRY thesis by transplanting red algal rubiscos with enhanced kinetics will diatom species have been reported, suggesting the requirement for need to take into account the requirement for a compatible Rca. an activase (23, 24). Understanding and defining the activase re-

+ quirement of eukaryotic red-type rubiscosisespeciallypertinent, rubisco | activase | photosynthesis | AAA proteins | red algae because a number of these have been demonstrated to possess kinetic properties (such has high CO2/O2 specificity factors) n all photosynthetic eukaryotes the enzyme ribulose 1,5-bisphos- that would confer enhanced photosynthetic properties to land Iphate carboxylase/oxygenase (rubisco) catalyzes the incorporation – – of carbon dioxide into biomass during the Calvin Benson Bassham Significance cycle (1). The majority of these organisms possess the form I-type enzyme, which forms an oligomer of large and small subunits in an L S stoichiometry. Form I rubiscos are phylogenetically deeply Eukaryotic phytoplankton of the red plastid lineage domi- 8 8 nate the oceans and are responsible for a significant pro- divided between a green-type clade (forms IA and IB) derived from portion of global photosynthetic CO fixation. In contrast to cyanobacteria and a red-type clade (forms IC and ID) of proteo- 2 their ecological importance, relatively little is known about bacterial origin (2, 3). Eukaryotic phytoplankton of the red plastid the biochemical properties of their carbon dioxide fixation lineage all contain the red-type form ID enzyme and dominate the machinery. In plants, the carbon dioxide fixing enzyme ribulose modern oceans (4). The geochemical importance of these organ- 1,5-bisphosphate carboxylase/oxygenase (rubisco) forms inactive isms is enormous, with diatoms alone believed to be responsible for ∼ complexes with sugar phosphates and needs to be constantly 20% of global net primary productivity (5). remodeled by the motor protein rubisco activase (Rca). Here Rubisco has long been a target of crop improvement strategies we show that in red algae, rubisco also forms inhibited com- (6) due to its low catalytic efficiency in addition to its tendency to plexes, which can be rescued by a convergently evolved Rca catalyze abortive side reactions that result in damaged metabolites (7). similar to one described in photosynthetic bacteria. Some red One such compound is the oxygenation product 2-phosphoglycolate algal rubiscos are a target for crop improvement strategies, be- that needs to be repaired via photorespiration (8) and rubisco cause their kinetics are better suited to the current atmospheric inhibitors such as xylulose 1,5-bisphosphate (XuBP) that are then gas composition. dephosphorylated by specific phosphatases (9, 10). XuBP, other sugar phosphates, and even rubisco’s bona fide substrate ribulose Author contributions: N.L. and O.M.-C. designed research; N.L. and Y.-C.C.T. performed 1,5-bisphosphate (RuBP) can tightly bind to the active site (11), research; N.L. analyzed data; and O.M.-C. wrote the paper. resulting in dead-end complexes that need to be reactivated for The authors declare no conflict of interest. photosynthetic CO2 fixation to proceed. Conformational remod- This article is a PNAS Direct Submission. eling of dead-end complexes, which results in release of the in- 1To whom correspondence should be addressed. Email: [email protected]. hibitor, is achieved in diverse organisms by a growing group of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. molecular chaperones known as the rubisco activases (Rcas) (12). 1073/pnas.1610758113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1610758113 PNAS | December 6, 2016 | vol. 113 | no. 49 | 14019–14024 Downloaded by guest on October 1, 2021 plants if successfully expressed (21, 25). Currently these efforts are A B void 440 158 kDa hampered by an incomplete appreciation of their biogenesis re- quirements (3, 26). kDa 50 Here we demonstrate that under physiological temperatures 40 Cyanidioschyzon merolae the red algal rubisco from forms tightly 30 inhibited complexes that can be activated by its cognate activase, 25 which is a heterooligomeric complex consisting of the gene products of nuclear- and plastid-encoded cbbX. By analyzing a CmN CmP series of mutant activases, we show that the two isoforms func- C RsRca CmNP tion in a highly coordinated manner, and that the nuclear- activase CmN (CmNP) 2+ encoded subunit plays a more critical role in activase function. RuBP CO2 + Mg Translational photosynthesis approaches that aim to take ad- CmP vantage of red algal rubisco kinetics will need to take into ac- ER E ECM CmNP count the requirement of a compatible Rca. inhibited inactive active RuBP 2+ CO2 + Mg Results D E The Red Algal Rca Functions as a Heterooligomer. A sequence alignment of selected prokaryotic and nuclear- and plastid- encoded cbbX sequences indicated that residues that have been demonstrated to be functionally important in the bacterial acti- vase from Rhodobacter sphaeroides (RsRca) are in general highly conserved (Fig. S1). Bioinformatic analyses have previously shown that cbbX sequences can be grouped in three clusters: prokaryotic, plastid encoded, and nuclear (or nucleomorph) encoded (Fig. S2) (27, 20, 28). The prokaryotic sequences are more closely related to the plastid genes (∼70% aa identity), whereas the nuclear genes have diverged more extensively (∼50% aa identity to the other groups). CmN CmP To understand the significance of the distribution of Rca CmNP RsRca genes observed in red lineage phytoplankton, we decided to CmN+CmP recombinantly produce in Escherichia coli and biochemically characterize the gene products of both nuclear- and plastid- Fig. 1. The red algal Rca functions as a heterooligomer. (A) SDS/PAGE analysis of purified activase proteins. A total of 3 μg protein was loaded per encoded cbbX isoforms from the red algal model organism C. merolae lane. (B) Gel-filtration analysis of the algal activase proteins. At identical (29). The construct encoding the nuclear isoform (CmN) loading concentration (20 μg) the heterooligomer CmNP elutes as a larger comprised residues 95–401,basedonsequencealignmenttothe complex than the individual isoforms. Buffer conditions were as follows: functional bacterial activase RsRca and the plastid-encoded CmN and CmNP, buffer A; CmP, buffer A adjusted to pH = 10, 300 mM NaCl. isoform CmP (Fig. S1). These isoforms have been produced in (C) Rcas catalyze the removal of inhibitory RuBP from inhibited active sites E. coli previously, but were not tested for activase function (30). (ER) to form the inactive apo-enzyme E. E spontaneously binds the cofactors 2+ Following overexpression in E. coli, CmN was easily obtained at CO2 and Mg to form the functional holoenzyme ECM. (D) The hetero- high purity by using a combination of affinity, anion-exchange, oligomer, but not individual isoforms, can activate R. sphaeroides rubisco in and size-exclusion chromatography (Fig. 1A and Fig. S3A). In the inhibited form. Rubisco activity assay of active (RsECM) and inhibited (RsER) rubisco complexes (0.5 μM active sites) was performed in the presence contrast, in our hands, purification of the plastid isoform (CmP) μ = and absence of C. merolae activase complexes (5 M activase protomer). Assay was only possible at pH 10, while maintaining high salt con- temperature was 25 °C. (E) The ATPase activity of the heterooligomer, but not centrations (300 mM NaCl) to avoid protein aggregation (Fig. individual isoforms, is stimulated by RuBP and inhibited R. sphaeroides rubisco. S3B). However, when cell pellets of E. coli producing CmN and ATPase assays of the activases (5 μM protomer) performed at 25 °C in the CmP were mixed before lysis, the two proteins copurified during presence and absence of RuBP (1 mM) and R. sphaeroides ER (3 μMactivesites). anion exchange and gel-filtration chromatography (at pH = 8), Error bars indicate the mean and SD of at least three independent experiments. and aggregation of CmP was not observed. This suggested that CmN + CmP indicates the heterooligomeric complex reconstituted from plastid- and nuclear-encoded isoforms formed a hetero- purified CmN and CmP, whereas CmNP is the copurified complex. oligomeric complex (CmNP) (Fig. 1A and Fig. S3C). As expec- ted, coexpression of CmN and CmP in a strain harboring a is a hexamer consisting of three CmN and three CmP subunits, pETDuet plasmid encoding both isoforms also allowed purifi- D whereas the homooligomeric proteins occupy a mixture of smaller cation of CmNP (Fig. S3 ). In this case, only CmP was fused to a B N-terminal His –ubiquitin tag for affinity purification, providing oligomeric states (Fig. 1 ). 6 Next we tested whether the algal activase complexes were additional evidence for complex formation. Densitometric R. sphaeroides analysis of the coexpressed CmNP complex was consistent with a functional in activating the red-type rubisco from . E To carboxylate RuBP, rubisco active sites need to first bind two 1:1 stoichiometry (Fig. S3 ). Gel-filtration analysis of the puri- 2+ cofactors: a nonsubstrate CO2 and a Mg ion (31, 32). This fied complexes indicated that both CmN and CmP formed 2+ polydisperse, concentration-dependent oligomers (Fig. S4 A and binding results in the activated holoenzyme [enzyme-CO2-Mg B). In contrast, CmNP presented a concentration-independent (ECM)]. In the absence of the cofactors, active sites can bind monodisperse oligomeric state (albeit with a slight trailing RuBP instead of forming the inhibited complex [enzyme-RuBP shoulder), when applied at concentrations of 0.4 mg/mL (cor- (ER)] (33). Rubisco activases conformationally remodel rubisco responding to 20 μg protein loaded) or higher (Fig. 1B and Fig. to convert ER to the apo form E (Fig. 1C). Here, using the S4C). The elution volume of CmNP corresponded to a Stokes spectrophotometric rubisco assay (34), we compare the carbox- radius of 5.8 nm (Fig. S4D), within 10% of the value measured ylation activities of ECM and ER complexes in the presence or + for the hexameric AAA protein AfQ2 (5.4 nm) (15). In sum- absence of the activase. mary, our data are consistent with the notion that at an equal Both CmN and CmP were nonfunctional, but the heterooligomer loading concentration of 0.4 mg/mL, the heterooligomeric CmNP displayed robust activase activity (Fig. 1D), corresponding to ∼50%

14020 | www.pnas.org/cgi/doi/10.1073/pnas.1610758113 Loganathan et al. Downloaded by guest on October 1, 2021 of RsRca activase function when activase concentrations were A B limiting (Fig. S4E). Reconstituting CmNP from purified CmN and CmP also allowed formation of the functional activase (Fig. 1D). The bacterial red-type activase RsRca displays ATPase activity only in the presence of the allosteric regulator ribulose 1,5-bisphosphate (RuBP), and the activity is strongly stimulated by its substrate rubisco (Fig. 1E) (14). No ATPase activity could be detected for CmN and CmP in isolation, whereas both copurified and recon- stituted CmNP displayed a low basal ATPase activity that could be stimulated by RuBP and inhibited rubisco. Strikingly the observed catalytic rate for CmNP was only a fraction (10–20%) of that measured for the bacterial activase RsRca under equivalent con- ditions (Fig. 1E). Finally we tested the ability of RsRca and CmNP to remove the nonphysiological, tight binding transition state analog C D 2-carboxy-arabinitol 1,5 bisphosphate (CABP) (35) from the ECM holoenzyme (Fig. S4F). Both activases were able to rapidly activate ECM-CABP (ECMC) complexes (Fig. S4G). In comparison, green- type Rca from spinach is unable to dissociate CABP from its cognate rubisco (36), whereas the CbbQO activases from chemoautotrophic bacteria are able to do so (15).

Characterization of the Red Algal Rubisco Activation System. Having established the identity of the functional algal Rca, we aimed to reproduce the entire algal activation system. As eukaryotic rubis- * cos have still not been successfully produced recombinantly, we RsRca CmNP CmNP purified the C. merolae rubisco from algal culture to homogeneity 25 °C 25 °C 45 °C using anion exchange chromatography and gel filtration (Fig. S5A). Consistent with earlier work using the rubisco from another Fig. 2. Biochemical characterization of the red-algal rubisco activation red algal thermophile G. sulphuraria (22), we found that C. mer- system. (A and B) The algal CmNP activase can efficiently activate inhibited olae rubisco did not form stably inhibited complexes with RuBP or algal ER (A) and ECMC (B) complexes at physiological temperature. Rubisco B activity assay conditions were as follows: 45 °C, 0.5 μM rubisco active sites CABP at 25 °C (Fig. S5 ). We therefore decided to investigate the and 5 μM activase protomer. (C) Regulation of the ATPase activity of bac- properties of this enzyme at the more physiologically relevant terial and algal activases (5 μM protomer) by RuBP (1 mM) and inhibited temperature of 45 °C (29). ECM formation was efficient at both bacterial and algal rubisco complexes (3 μM active sites). *, not measured temperatures with the holoenzyme displaying linear activity con- due to RsER instability at 45 °C. (D) The algal activase is thermostable. ATPase − − sistent with a carboxylation velocity of 0.4·s 1 at 25 °C and 0.7·s 1 assays of bacterial and algal activases (5 μM protomer) were performed at at 45 °C. At the higher temperature, incubation of rubisco with temperatures up to 55 °C in the presence of 1 mM RuBP. Error bars indicate BIOCHEMISTRY either inhibitor readily led to the formation of stably inhibited the mean and SD of at least three independent experiments. complexes that did not spontaneously reactivate at 25 °C (Fig. S5 C and D). At 45 °C, the CmNP activase was able to remove both inhibitors from the inhibited red-algal rubisco complexes with plant Rca (37), thermostable activases are of potential biotech- complete activation of CmER achieved after 2 min (Fig. 2 A and nological interest (38). B). At 25 °C, activation of inhibited algal rubisco by CmNP was The Role of the Allosteric Regulator RuBP. The nucleotide-bound marginal (Fig. S5 C and D), in stark contrast to its robust ability to bacterial RsRca undergoes an oligomeric transition from an activate the bacterial enzyme at this temperature (Fig. 1D). At ATPase inactive fibril to the functional hexamer, which is trig- 35 °C (but not at 25 °C) the simpler homooligomeric RsRca was gered by the binding of the allosteric regulator RuBP and provides able to activate inhibited algal CmER, but displayed low activity – when exposed to CmECMC (Fig. S5 E and F). At 45 °C, ap- a regulatory mechanism to respond to increases in Calvin Benson proximately one CmNP hexamer per inhibited active site was re- cycle flux (14). These transitions were earlier demonstrated using quired to achieve full activation of CmER (Fig. S6 A and B). At negative-stain electron microscopy, but we found they can be re- capitulated using gel filtration (Fig. 3A). In the Apo form, RsRca subsaturating CmNP concentrations, rubisco activity became lin- ∼ ear, corresponding to a fractional activity of ECM, indicating that behaved as a large 400-kDa polydisperse oligomer. In buffer activation and inhibition by RuBP was proceeding at equivalent containing Mg-ATP, RsRca eluted close to the void volume, rates (Fig. S6 A and B). When CmNP concentrations were satu- consistent with previously observed fibrils. In contrast, in the rating, full activation could be achieved even when rubisco active presence of both RuBP and MgATP, the condition under which site concentrations were as low as 0.05 μM(Fig. S6C). hexamers were previously observed, a monodisperse symmetrical When comparing the relative ability of different rubisco peak at 1.23 mL was obtained. In contrast, under equivalent complexes to stimulate ATPase activity, we noticed that inhibi- conditions, CmNP eluted as a species resembling the RsRca ted algal complexes did not stimulate RsRca ATPase at 25 °C, hexamer (although a shoulder was observed, indicating some level whereas both algal and bacterial complexes resulted in modest, of subunit dissociation) (Fig. 3B). Hence, although the allosteric but concentration-dependent stimulation of CmNP at both 25 °C activation of ATPase activity by RuBP is conserved in the algal and 45 °C (Fig. 2C and Fig. S6 D and E). Intrigued by the activase, the associated oligomeric transition from fibril to hex- temperature dependence of the algal rubisco activation system, amer has apparently not been retained during evolution. Kinetic we determined the optimal temperature of CmNP for ATP hy- analysis of CmNP ATP hydrolysis was consistent with a non- drolysis to be ≥55 °C (Fig. 2D). CmNP lost activity upon in- cooperative model in the absence of RuBP. In the presence of the cubation at 60 °C and was completely denatured at 65 °C (Fig. ligand the substrate concentration required for half-maximal ac- S6F). The bacterial RsRca exhibited a thermal optimum of 35 °C tivity was reduced by 75%, and the maximal catalytic rate almost for its ATPase function, and the protein aggregated at higher doubled (Fig. S6G). Importantly, the data were not consistent with temperatures (Fig. 2D). Given the thermolabile nature of crop the Michaelis–Menten model, but indicated positive cooperativity

Loganathan et al. PNAS | December 6, 2016 | vol. 113 | no. 49 | 14021 Downloaded by guest on October 1, 2021 strongly stimulate CmN ATPase activity, a phenomenon seen ABvoid 440 158 kDa void 440 158 kDa RsRca CmNP previously in ClpB (45). Unexpectedly, ATPase (but not activase) activity of a complex containing a arginine finger substitution in CmN could be restored by a concomitant Walker B mutation in CmP. The resultant complex CmNRPB had a high basal ATPase rate equivalent to that of CmNPB, which however was not stimulated further by RuBP or inhibited rubisco (Fig. 4B). In contrast, the inverse arrangement CmNBPR was nonfunctional (Fig. 4B). We conclude that subunits in the heterohexamer are arranged in an alternating sequence, which in complex CmNRPB permits the formation of three func- tional ATPase sites harbored by the nuclear-encoded subunit. These are formed by the Walker motifs of CmN and the arginine Fig. 3. The bacterial, but not the red-algal activases undergo RuBP-triggered A B oligomeric transitions. Gel-filtration analysis of 20 μg of bacterial (A) and algal finger of CmP (Fig. 4 and ). An alternative dimer of trimer (B) activase protein in buffer A supplemented with 0.1 mM RuBP, 1 mM ATP, arrangement would lead to only a single functional active site per

and 5 mM MgCl2 as indicated. hexamer (44), and is not consistent with this result. The other + described heterohexameric AAA systems Yta10/12 and Pex1/6 also follow a strictly alternating sequence (42–44). with a Hill constant of 2.1. Hence for the algal activase binding of the allosteric regulator, RuBP results in conformational changes Both Subunit Types Are Involved in Rubisco Remodeling. Similar to + that lead to higher ATP hydrolysis efficiency and introduce other AAA ATPases (17, 46), the mechanism of bacterial RsRca cooperativity, but the ligand does not trigger oligomeric transitions uses an axial pore loop 1 tyrosine to transiently thread the C as seen for the bacterial homolog. Positive cooperativity has re- terminus of the rubisco large subunit to trigger the release of cently also been described for the green-type Rca from tobacco, bound inhibitors. We tested the ability of CmNP to activate where it was induced both by the presence of the competitive R. sphaeroides 2+ rubisco mutants with two- or four-amino acid deletions inhibitor ADP or excess Mg (39). at the C terminus of the large subunit (Fig. 5A). As observed for CmN Subunits Are More Critical than CmP for ATPase and Activase Function. Given the strict requirement for both nuclear- and plastid-encoded subunits in the functional red algal activase, we A decided to ask whether the subunits had evolved unique roles or RuBP whether both contributed equally to their function of ATP hydrolysis and rubisco activation. Toward this end, we constructed a series of R239 aminoacidexchangesinbothsubunits(primerslistedinTable S1) ADP and, by mixing cell pellets expressing the desired CmN and CmP variants, purified CmNP complexes harboring different combina- R194 tions of mutations. All variant enzymes examined here could be K80 purified as the assembled heterohexamer (Fig. S7 A and B). E138 Initially we considered the Rca and ATPase activity of mutants containing substitutions in catalytic residues of the Walker motifs and arginine fingers of their nucleotide binding domains B (NBDs). The Walker motifs interact with the nucleotide bound to its NBD, and the arginine finger interacts with the γ-phos- CmP phate of the nucleotide bound to the adjacent active site (40) A (Fig. 4 ). Both single and double arginine finger substitutions CmN RB R B B R (R194A: residue numbering follows RsRca, Fig. S1) abolished R B ATPase and activase activity, irrespective of subunit (Fig. 4B). BRBR Similarly, a Lys-to-Ala (K80A) substitution in the Walker A motif that generally abolishes nucleotide binding (17) led to completely inactive ATPase and activase complexes in all in- R B B R R B C R P stances (Fig. S7 ). These results indicated that the subunits do B P B P R P R P NP N NP B P not operate independently as proposed for ClpX (41), but are N NP N N N N instead stringently functionally coupled. In contrast to the Activase 100 ± 7 <1 72 ± 2 <1 <1 <1 <1 <1 <1 complete loss of functionality observed for Walker A and argi- (% WT) nine finger substitutions, we found that a mutation of the Walker Fig. 4. The subunits are highly coordinated and arranged alternatingly. B glutamate (E138Q) was largely tolerated by CmP alone (A) Active site architecture based on the atomic model of the RsRca hexamer (maintaining 72% of activase activity), but abolished ATPase [Protein Data Bank (PDB) ID code 3ZUH]. (B) Biochemical characterization of and activase activity when present in either CmN or both sub- CmNP active site variants. Superscripts indicate the following amino acid units (Fig. 4B and Fig. S7D). This result is reminiscent of those substitutions: B, E138Q (Walker B) and R, R194A (arginine finger). The Inset + observed for two other heterooligomeric AAA proteins, Pex1/6 schematic illustrates the alternative arrangement of subunits consistent with (42, 43) and Yta10/12 (44), where a Walker B mutation in one formation of three functional ATPase active sites (black letters, motifs intact R B subunit, but not the other, abolished functionality of the com- and red letters, mutated) for the N P variant. ATPase assays were per- plex. In contrast to those systems, CmNPB complexes hydrolyzed formed at 45 °C using equivalent conditions to those in Fig. 2C. In the rubisco activity assays, red numbers indicate no detectable activity under conditions ATP at around twice the rate of CmNP in both the absence and as in Fig. 2A. Green numbers indicate relative initial rate of ECM formation presence of the regulator RuBP. The Walker B substitution used was quantified in assays containing 0.3 μM active sites (CmER) and 1 μM here generally leads to an active site that binds but does not activase protomer at 45 °C (Fig. S7D). Error bars indicate the mean and SD of hydrolyze ATP (17). Nucleotide-bound CmP thus appears to at least three independent experiments.

14022 | www.pnas.org/cgi/doi/10.1073/pnas.1610758113 Loganathan et al. Downloaded by guest on October 1, 2021 A RsΔ4 RsΔ2 nuclear subunit plays a more critical (but not exclusive) role in remodeling inhibited rubisco complexes. To further understand the requirement for binding of the al- RsRbcL losteric regulator, we characterized variants mutated at the RuBP CmRbcL binding site. All residues shown to be involved in RuBP binding in RsRca (14) are conserved in both CmN and CmP (Fig. S1), so we decided to analyze the effect of substituting Arg-239 with alanine. B C This residue is predicted to coordinate a phosphate moiety of RuBP. When present in either the nuclear (CmNSP) or both isoforms (CmNSPS), both ATPase and Rca activity were com- pletely abolished (Fig. 5D). CmNPS retained ∼50% basal ATPase activity, which was no longer stimulated by RuBP or rubisco. Nevertheless this complex had 13% of the wild-type Rca activity (Fig. 5D). The phenotype of these variants is consistent with the notion that allosteric RuBP binding to the main catalytic subunit CmN of the algal Rca is essential for activase function.

RsER RsΔ2ER RsΔ4ER Discussion D CmP It has been unclear whether the propensity of rubisco to form dead-end inhibited complexes and the requirement for an acti- S vase to remodel these extends into the red-lineage eukaryotic CmN Y Y phytoplankton (22). Here we provide unambiguous biochemical evidence that red algal form ID rubisco forms stably inhibited S complexes at physiological temperatures that can be rescued by its cognate activase CmNP. The activase is a 1:1 heterohexamer of plastid- and nuclear-encoded subunits. In accordance with observed gene distributions, we expect this observation to hold widely for organisms of the red plastid lineage, which includes diatoms, brown algae, and cryptophytes. It is noteworthy that the algal activase overall was highly functional in remodeling bac- terial rubisco even at suboptimal temperatures (Fig. 1D and Fig. S4E), whereas RsRca exhibited relatively less activity (and a lack S S Y Y S P Y P S P Y P of ATPase stimulation) with algal rubisco as the substrate (Fig. NP N N NP N NP N S5 C–F and Fig. 2C). It remains to be established whether these

100 ± 13 13 ± 1 <1 <1 19 ± 2 <1 <1 results are caused by differences in relative rubisco complex stability, activase efficiency, protein–protein interaction com- BIOCHEMISTRY Activase patibility, or a combination of these factors. (% WT) RuBP binding (S) Pore loop (Y) Although the algal activase has conserved its dependence on pore Fig. 5. The mechanism of the algal activase is conserved. (A) Alignment of the loop 1 tyrosines and the rubisco large subunit C terminus for rubisco rubisco large subunit C termini of R. sphaeroides and C. merolae.(B) Impaired remodeling activity (Fig. 5 B and D), and thus uses the same activation of R. sphaeroides rubisco with two or four amino acids truncated mechanism as the bacterial activase, its regulatory properties differ from the large subunit. Assay conditions were as described in Fig. 1D and the markedly (Fig. S8). Whereas RsRca forms ATPase inactive fibrils in relative rate of ECM formation was quantified. (C)TruncatedR. sphaeroides the absence of the allosteric regulator RuBP, the algal enzyme has a rubisco does not stimulate the ATPase activity of the activases. Assays were basal constitutive ATPase rate and forms the hexamer under the performedasinFig.1E in the presence of 1 mM RuBP. (D) Biochemical char- same conditions. In RsRca, RuBP triggers an oligomeric transition acterization of CmNP variants mutated at the putative RuBP binding site and pore loop 1. Superscripts indicate the following amino acid substitutions: S, to the ATPase active hexamer, whereas the catalytic efficiency for R239A (sugar binding) and Y, Y114A (pore loop tyrosine). Experiments were ATP hydrolysis increases for CmNP. Once the inhibited rubisco performed as in Fig. 4B but using 2 μM activase protomer to quantify activase complex is engaged, ATPase activity of RsRca increases severalfold activity (green numbers). Error bars indicate the mean and SD of at least three and in comparison is only modestly stimulated in the algal enzyme independent experiments. (Fig. S8). We predict that characterization of more red-type acti- vases will uncover more diverse regulatory behavior that will guide the choice of activases to be used in biotechnological ventures RsRca earlier (14), both activation (Fig. 5B) and ATPase stim- aiming to engineer photosynthetic CO2 fixation. ulation of CmNP was impaired (Fig. 5C), supporting a conserved Our characterization of activase variants mutated in key mechanism. functional residues suggests an alternating arrangement of sub- We therefore decided to probe the relative importance of the units with a catalytically dominant CmN (Fig. S8). We interpret key pore loop tyrosine in the two isoforms. Pore loop tyrosine the high activase functionality of CmNPB (Fig. 4B) to indicate (Y114A) substitutions led to complete abolishment of activase that in the wild type the CmP subunit is either catalytically inert activity, when present in the nuclear or both subunits (CmNYP or its ATP hydrolysis function does not significantly contribute to and CmNYPY)(Fig.5D). These complexes displayed increased the energy used during rubisco remodeling. Despite its relegated basal and RuBP-stimulated ATPase activity, likely due to de- role in ATP hydrolysis, the phenotype of Walker A and arginine finger variants suggests CmP plays a critical role in tightly co- creased steric constraints of pore loop movement as observed for Y ordinating ATP hydrolysis. ClpX (46). The CmNP variant displayed wild-type ATPase ac- The availability of sensitive and rapid biochemical assays for tivity (although stimulation by inhibited rubisco was reduced) and both ATPase and activase function makes rubisco activation a retained 19% of activase activity. These results confirm the im- highly accessible system to study structure–function relationships + portance of the axial pores in the remodeling mechanism of the of evolutionarily diverse AAA proteins. At the same time, ef- algal activase, and similar to the catalytic variants, suggest that the forts to engineer photosynthesis, in particular the rate-limiting

Loganathan et al. PNAS | December 6, 2016 | vol. 113 | no. 49 | 14023 Downloaded by guest on October 1, 2021 CO2-fixing reactions, are gaining momentum (47). To effectively and were purified as described in SI Materials and Methods. Rubisco and ATPase efficiently harness the potential of , it is essential to activity was measured using coupled spectrophotometric assays (34, 49). Inhibited rubisco complexes were prepared as described in SI Materials carefully characterize the diversity of CO2-fixation modules that are found in the vast treasure troves of metagenomic studies (48). and Methods. Materials and Methods ACKNOWLEDGMENTS. We thank Dr. Spencer Whitney (Australian National University) for the gift of CPBP and Melvin Chua, Maria Claribel Lapina, and Detailed experimental procedures are described in SI Materials and Methods. Lynette Liew for technical assistance. The research was funded by a Nanyang All activase proteins and R. sphaeroides rubisco were produced recombi- Technological University startup grant and Ministry of Education of Singa- nantly in E. coli. C. merolae rubisco was obtained from algal culture. Proteins pore AcRF Tier2 Grant MOE2013-T2-2-089 (to O.M.-C.).

1. Andrews TJ, Lorimer GH (1987) Rubisco: Structure, mechanisms, and prospects for im- 29. Matsuzaki M, et al. (2004) Genome sequence of the ultrasmall unicellular red alga provement. The Biochemistry of Plants: A Comprehensive Treatise, Vol. 10, Photosynthesis, Cyanidioschyzon merolae 10D. Nature 428(6983):653–657. eds Hatch MD, Boardman NK (Academic, New York), pp 131–218. 30. Fujita K, Tanaka K, Sadaie Y, Ohta N (2008) Functional analysis of the plastid and 2. Tabita FR (1999) Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: A dif- nuclear encoded CbbX proteins of Cyanidioschyzon merolae. Genes Genet Syst 83(2): ferent perspective. Photosynth Res 60(1):1–28. 135–142. 3. Hauser T, Popilka L, Hartl FU, Hayer-Hartl M (2015) Role of auxiliary proteins in Rubisco 31. Cleland WW, Andrews TJ, Gutteridge S, Hartman FC, Lorimer GH (1998) Mechanism of biogenesis and function. Nat Plants 1:15065. Rubisco: The carbamate as general base. Chem Rev 98(2):549–562. 4. Falkowski PG, et al. (2004) The evolution of modern eukaryotic phytoplankton. 32. Lorimer GH, Badger MR, Andrews TJ (1976) The activation of ribulose-1,5-bisphosphate – Science 305(5682):354 360. carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested 5. Nelson DM, Treguer P, Brzezinski MA, Leynaert A, Queguiner B (1995) Production and mechanism, and physiological implications. Biochemistry 15(3):529–536. dissolution of biogenic silica in the ocean: Revised global estimates, comparison with 33. Jordan DB, Chollet R (1983) Inhibition of ribulose bisphosphate carboxylase by sub- regional data and relationship to biogenic sedimentation. Global Biogeochem Cycles strate ribulose 1,5-bisphosphate. J Biol Chem 258(22):13752–13758. – 9(3):359 372. 34. Kubien DS, Brown CM, Kane HJ (2011) Quantifying the amount and activity of Rubisco in 6. Parry MA, et al. (2013) Rubisco activity and regulation as targets for crop improve- leaves. Methods Mol Biol 684:349–362. ment. J Exp Bot 64(3):717–730. 35. Pierce J, Tolbert NE, Barker R (1980) Interaction of ribulosebisphosphate carboxylase/ 7. Linster CL, Van Schaftingen E, Hanson AD (2013) Metabolite damage and its repair or oxygenase with transition-state analogues. Biochemistry 19(5):934–942. pre-emption. Nat Chem Biol 9(2):72–80. 36. Robinson SP, Portis AR (1988) Release of the nocturnal inhibitor, carboxyarabinitol-1- 8. Bauwe H (2011) Photorespiration: The Bridge to C4 photosynthesis. C4 Photosynthesis phosphate, from ribulose bisphosphate carboxylase oxygenase by rubisco activase. and Related CO2 Concentrating Mechanisms, Chapter 6, eds Raghavendra SA, Sage FR FEBS Lett 233(2):413–416. (Springer, The Netherlands), pp 81–108. 37. Scafaro AP, et al. (2016) Heat tolerance in a wild Oryza species is attributed to 9. Bracher A, Sharma A, Starling-Windhof A, Hartl FU, Hayer-Hartl M (2015) Degradation maintenance of Rubisco activation by a thermally stable Rubisco activase ortholog. of potent Rubisco inhibitor by selective sugar phosphatase. Nat Plants 1(1):14002. – 10. Andralojc PJ, et al. (2012) 2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase: New Phytol 211(3):899 911. Evidence for a wider role in plant Rubisco regulation. Biochem J 442(3):733–742. 38. Kurek I, et al. (2007) Enhanced thermostability of Arabidopsis Rubisco activase im- 11. Parry MAJ, Keys AJ, Madgwick PJ, Carmo-Silva AE, Andralojc PJ (2008) Rubisco reg- proves photosynthesis and growth rates under moderate heat stress. Plant Cell 19(10): – ulation: A role for inhibitors. J Exp Bot 59(7):1569–1580. 3230 3241. 12. Mueller-Cajar O, Stotz M, Bracher A (2014) Maintaining photosynthetic CO2 fixation 39. Hazra S, Henderson JN, Liles K, Hilton MT, Wachter RM (2015) Regulation of ribulose- via protein remodelling: The Rubisco activases. Photosynth Res 119(1–2):191–201. 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase: Product inhibition, coop- 13. Salvucci ME, Portis AR, Jr, Ogren WL (1985) A soluble chloroplast protein catalyzes erativity, and magnesium activation. J Biol Chem 290(40):24222–24236. ribulosebisphosphate carboxylase/oxygenase activation in vivo. Photosynth Res 7(2): 40. Wendler P, Ciniawsky S, Kock M, Kube S (2012) Structure and function of the AAA+ 193–201. nucleotide binding pocket. Biochim Biophys Acta 1823(1):2–14. 14. Mueller-Cajar O, et al. (2011) Structure and function of the AAA+ protein CbbX, a 41. Martin A, Baker TA, Sauer RT (2005) Rebuilt AAA + motors reveal operating principles red-type Rubisco activase. Nature 479(7372):194–199. for ATP-fuelled machines. Nature 437(7062):1115–1120. 15. Tsai YC, Lapina MC, Bhushan S, Mueller-Cajar O (2015) Identification and charac- 42. Ciniawsky S, et al. (2015) Molecular snapshots of the Pex1/6 AAA+ complex in action. terization of multiple rubisco activases in chemoautotrophic bacteria. Nat Commun Nat Commun 6:7331. 6:8883. 43. Gardner BM, Chowdhury S, Lander GC, Martin A (2015) The Pex1/Pex6 complex is a 16. Portis AR, Jr (2003) Rubisco activase: Rubisco’s catalytic chaperone. Photosynth Res heterohexameric AAA+ motor with alternating and highly coordinated subunits. 75(1):11–27. J Mol Biol 427(6 Pt B):1375–1388. 17. Hanson PI, Whiteheart SW (2005) AAA+ proteins: Have engine, will work. Nat Rev 44. Augustin S, et al. (2009) An intersubunit signaling network coordinates ATP hydrolysis Mol Cell Biol 6(7):519–529. by m-AAA proteases. Mol Cell 35(5):574–585. 18. Maier U-G, Fraunholz M, Zauner S, Penny S, Douglas S (2000) A nucleomorph-encoded 45. Zeymer C, Fischer S, Reinstein J (2014) trans-Acting arginine residues in the AAA+ – CbbX and the phylogeny of RuBisCo regulators. Mol Biol Evol 17(4):576 583. chaperone ClpB allosterically regulate the activity through inter- and intradomain 19. Delwiche CF, Palmer JD (1996) Rampant horizontal transfer and duplication of rubisco communication. J Biol Chem 289(47):32965–32976. – genes in eubacteria and plastids. Mol Biol Evol 13(6):873 882. 46. Martin A, Baker TA, Sauer RT (2008) Pore loops of the AAA+ ClpX machine grip 20. Hovde BT, et al. (2015) Genome sequence and transcriptome analyses of Chrys- substrates to drive translocation and unfolding. Nat Struct Mol Biol 15(11):1147–1151. ochromulina tobin: Metabolic tools for enhanced algal fitness in the prominent order 47. Lin MT, Occhialini A, Andralojc PJ, Parry MA, Hanson MR (2014) A faster Rubisco with Prymnesiales (Haptophyceae). PLoS Genet 11(9):e1005469. potential to increase photosynthesis in crops. Nature 513(7519):547–550. 21. Read BA, Tabita FR (1994) High substrate specificity factor ribulose bisphosphate 48. Sunagawa S, et al.; Tara Oceans coordinators (2015) Ocean plankton. Structure and carboxylase/oxygenase from eukaryotic marine algae and properties of recombinant function of the global ocean microbiome. Science 348(6237):1261359. cyanobacterial RubiSCO containing “algal” residue modifications. Arch Biochem Biophys 49. Stotz M, et al. (2011) Structure of green-type Rubisco activase from tobacco. Nat 312(1):210–218. Struct Mol Biol 18(12):1366–1370. 22. Pearce FG (2006) Catalytic by-product formation and ligand binding by ribulose bi- 50. Baker RT, et al. (2005) Using deubiquitylating enzymes as research tools. Methods sphosphate carboxylases from different phylogenies. Biochem J 399(3):525–534. Enzymol 398:540–554. 23. Young JN, et al. (2016) Large variation in the Rubisco kinetics of diatoms reveals di- 51. Ewalt KL, Hendrick JP, Houry WA, Hartl FU (1997) In vivo observation of polypeptide versity among their carbon-concentrating mechanisms. J Exp Bot 67(11):3445–3456. flux through the bacterial chaperonin system. Cell 90(3):491–500. 24. MacIntyre HL, Sharkey TD, Geider RJ (1997) Activation and deactivation of ribulose- 52. Minoda A, Sakagami R, Yagisawa F, Kuroiwa T, Tanaka K (2004) Improvement of 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in three marine microalgae. Photosynth Res 51(2):93–106. culture conditions and evidence for nuclear transformation by homologous re- 25. Andrews TJ, Whitney SM (2003) Manipulating ribulose bisphosphate carboxylase/ combination in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 45(6): – oxygenase in the chloroplasts of higher plants. Arch Biochem Biophys 414(2):159–169. 667 671. 26. Whitney SM, Baldet P, Hudson GS, Andrews TJ (2001) Form I Rubiscos from non-green 53. Lan Y, Mott KA (1991) Determination of apparent K(m) values for ribulose 1,5- algae are expressed abundantly but not assembled in tobacco chloroplasts. Plant J bisphosphate carboxylase/oxygenase (Rubisco) activase using the spectrophoto- 26(5):535–547. metric assay of rubisco activity. Plant Physiol 95(2):604–609. 27. Fujita K, Ehira S, Tanaka K, Asai K, Ohta N (2008) Molecular phylogeny and evolution 54. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic of the plastid and nuclear encoded cbbX genes in the unicellular red alga Cyanidio- Acids Res 16(22):10881–10890. schyzon merolae. Genes Genet Syst 83(2):127–133. 55. Gouet P, Courcelle E, Stuart DI, Métoz F (1999) ESPript: Analysis of multiple sequence 28. Starkenburg SR, et al. (2014) A pangenomic analysis of the Nannochloropsis organ- alignments in PostScript. Bioinformatics 15(4):305–308. ellar genomes reveals novel genetic variations in key metabolic genes. BMC Genomics 56. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evo- 15:212. lutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729.

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