Structural perturbations of Rhodopseudomonas palustris form II

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Structural perturbations of Rhodopseudomonas palustris form II RuBisCO mutant enzymes that affect CO2-fixation Sriram Satagopan, Justin A. North, Mark A. Arbing, Vanessa A. Varaljay, Sidney N. Haines, John A. Wildenthal, Kathryn M. Byerly, Annie Shin, and F. Robert Tabita Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00617 • Publication Date (Web): 28 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Biochemistry

Structural perturbations of Rhodopseudomonas palustris form II RuBisCO mutant enzymes that affect CO2-fixation Sriram Satagopan, †,‡ Justin A. North, †,‡ Mark A. Arbing, §,‡ Vanessa A. Varaljay, †,# Sidney N. Haines, † John A. Wildenthal, † Kathryn M. Byerly, † Annie Shin, § and F. Robert Tabita*,†

‡These

†Department

§UCLA-DOE

authors contributed equally to this work

of Microbiology, The Ohio State University, Columbus, OH 43210, USA.

Institute, University of California Los Angeles, Los Angeles, CA 90095, USA.

#Current

address: Air Force Research Laboratory, Wright-Patterson AFB, Ohio, USA

ACS Paragon Plus Environment

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Running title: Structure-function analysis of R. palustris form II mutant RuBisCOs

*Corresponding author: [email protected]; Tel. (614) 292-4297; Fax. (614) 292-6337.

ABSTRACT: The enzyme ribulose 1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO2 via the Calvin-Benson-Bassham (CBB) cycle has been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas

palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analog 2-carboxyarabinitol 1,5bisphosphate (CABP), greatly facilitating structure-function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues


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(Ile165 and Met331) and other conserved and semi-conserved residues near the enzyme’s active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme was accomplished that overcomes negative enzymatic functions. Structure-function analyses with negative- and suppressor-mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme’s quaternary structure that influenced partial reactions that constitute RuBisCO’s carboxylation mechanism. In particular, structural perturbations in an inter-subunit interface appear to affect CO2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation-negative mutants to support a previously described sulfur-salvage function, one that appears to solely rely on the enzyme’s ability to catalyze the enolization of a substrate analogous to RuBP.


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INTRODUCTION The catalytic activity of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (E.C. 4.1.1.39) accounts for the conversion of atmospheric CO2 to most of the organic carbon found on earth. Natural variants of bonafide RuBisCO enzymes all catalyze the fixation of gaseous CO2 onto the substrate RuBP, resulting in the formation of two molecules of 3phosphoglycerate (3PGA). This occurs as part of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway for carbon assimilation in plants, algae, and certain phototrophic and autotrophic bacteria.1 In addition, RuBisCO also participates in the proposed reductive hexulose phosphate pathway for carbon assimilation in various archaea,2 and in nucleotide monophosphate metabolism in other archaea and bacteria.3-5 RuBisCO employs a complex mechanism to convert RuBP and CO2 to 3PGA, involving four distinct steps and three intermediates.6 Active-site competition by molecular oxygen and a propensity to catalyze side reactions account for the enzyme’s promiscuity.7 Most bonafide RuBisCOs can be classified into four phylogenetic groups (forms I, II, III, and II/III hybrid) based on sequence and structural homologies.1,5 The RuBisCO superfamily also includes form IV genes, which encode proteins that assemble into RuBisCO-like proteins (RLPs). RLPs share structural similarities with RuBisCOs but catalyze other reactions distinct from RuBP carboxylation.1 With a sequence identity of approximately 30% across the different lineages, divergent RuBisCOs adopt characteristic oligomeric states composed of a single catalytic dimer of large (L) subunits or concatemers thereof [forms II, II/III, III & IV; (L2)n)], or hexadecameric complexes of large (L) and small (S) subunits (form I; L8S8).1,6-8 Strikingly, the alignment of RuBisCO and RLP sequences indicate a pattern of conservation among residues that


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are known to be involved in RuBP enolization by RuBisCO.9 In the case of Bacillus subtilis and Geobacillus kaustophilus YkrW/MtnW RLPs, which function in a salvage pathway for methionine, equivalent residues are implicated in enolization of substrate 2,3-diketo-5methylthiopentyl-1-phosphate.10-13 Conversely, the RuBisCO catalytic residues involved in the later steps of the CO2 fixation mechanism, i.e., carboxylation of the RuBP 2,3-ene-diol(ate) intermediate, account for most differences in the active-site residues among RuBisCOs and RLPs.9 Among the different RuBisCO structural variants, form I RuBisCOs, particularly those from crop plants, have been studied because of the interest in enhancing photosynthetic efficiency by engineering kinetically improved variants.14 However, since the isolation and characterization of the dimeric (L2) form II RuBisCO from Rhodospirillum rubrum,15-17 it has been a model enzyme for the elucidation of various aspects of the enzyme’s catalytic mechanism.18 Apart from CO2-fixation, the R. rubrum form II RuBisCO is also required for sulfur-salvage function under anaerobic conditions via a presently unresolved process.19-22 Anaerobic growth complementation studies with an R. rubrum RuBisCO-deletion strain revealed that all extant forms of bona fide RuBisCO, i.e. forms I, II, and III, as well as CO2-fixation compromised mutant RuBisCO enzymes are able to complement for the unknown function catalyzed by the R. rubrum RuBisCO during 5methylthioadenosine (MTA) metabolism.8,21-24 It has been suggested elsewhere that RuBisCO residues that are important for RuBP enolization may also participate in different reactions with related substrates as part of different sulfur salvage pathways. 12,25,26 In this study, we have attempted to further delineate the role of RuBisCO structural regions in the distinct steps comprising the CO2 fixation mechanism using the R. palustris form II RuBisCO, which is now amenable to structure-function studies.8 Unlike the R. rubrum enzyme, X-ray crystal structure determination of the R. palustris from II RuBisCO with transition-state


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analog 2-carboxyarabinitol-1,5-bisphosphate (CABP) has been achieved, and the close homology between the two enzymes (76% amino acid identity) enables direct comparison to previous R. rubrum studies.8,27 Several RuBisCO mutants were generated and their abilities to complement for CO2-dependent autotrophic growth were assessed in a RuBisCO-deletion strain of Rhodobacter capsulatus (strain SB I/II-). The competencies for catalyzing the enolization reaction were indirectly inferred from MTA-dependent growth complementation of a RuBisCO/RLP-deletion strain of R. rubrum (strain IR).21,23 These studies were further supported by the selection of a suppressor-mutant enzyme and X-ray structure determinations of mutant enzymes, which together assisted in correlating structure-function relationships. From these studies, we conclude that subtle structural changes at an inter-subunit interface were likely to affect CO2 addition and subsequent steps in the reaction mechanism but not affect the preceding step leading to 2,3-enediol(ate) formation from RuBP. Conversely, the active-site residues known to facilitate formation of the 2,3-enediol(ate) from RuBP were indeed identified as being essential for both CO2-fixation and metabolism of MTA under anaerobic conditions.

MATERIALS AND METHODS Bacterial strains and growth conditions. RuBisCO-deletion strains of R. capsulatus (strain SB I/II)8 and Ralstonia eutropha (strain H16ΔLS),28 and RuBisCO/RLP-deletion strain of R. rubrum (strain IR)21,23 were used for complementation studies with wild-type and mutant genes encoding R. palustris form II RuBisCO (Table 1).


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Table 1. Genotypes and phenotypes of bacterial strains used for RuBisCO complementations. Strain

Genotype

Phenotype and Use

R. rubrum IR 21

ΔcbbMa::KmR, b Δrlp c::GmR,d SmR,i, KmR, GmR; Test competency for RuBP enolization

R. eutropha H16ΔLS

ΔcbbLSCH

28

e,g::SpR,h

e,f

ΔcbbLSMP SpR; Test functional competency for RuBP carboxylation

R. capsulatus SB I/II-

ΔcbbLSe::Sp,R,h

KmR, SpR; Test functional

8

ΔcbbMa::KmR,b

competency for RuBP carboxylation

a

cbbM – Gene encoding form II RuBisCO large subunit; b KmR – Gene cassette conferring

Kanamycin resistance (KmR); c rlp – Gene encoding form IV RuBisCO-like protein (RLP); d

GmR – Gene cassette conferring Gentamicin resistance (GmR);

e

cbbLS – Genes

encoding form I RuBisCO large (L) and small (S) subunits; f CH – Chromosomal genes; g MP

– Megaplasmid genes;

h

SpR – Gene cassette conferring Spectinomycin resistance

(SpR); i SmR – Streptomycin resistant. All R. rubrum, R. capsulatus, and R. eutropha strains were initially grown in liquid or on solid-agar PYE medium (3 g/L peptone, 3 g/L yeast extract, 266 mg/L MgSO4·7H2O, 75 mg/L CaCl2·2H2O, 11.8 mg/L FeSO4·7H2O, 20 mg/L ethylenediaminetetraacetic acid, 1 ml/L Ormerod’s trace elements solution,29 1 mg/L thiamine, 1 mg/L nicotinic acid, 15 μg/L biotin) supplemented with appropriate antibiotics at 30°C, as performed previously.8,21,28-


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Escherichia coli strain Top10 was used as the host (Thermo Fisher Scientific) for

cloning and plasmid amplification. For recombinant protein synthesis, E. coli BL21(DE3) was utilized as described elsewhere.8 E. coli S17 was used as the host strain for plasmid mobilization.8,23,28 All E. coli strains were cultured aerobically in lysogeny broth (LB) at 37°C. For heterotrophic growth selection of transconjugants, PYE medium was utilized for R. capsulatus8 and R. rubrum,21,23 and Ormerod’s minimal medium with 0.4 % w/v malate was used for R. eutropha,28-31 with supplemented antibiotics: 25 μg/ml kanamycin, 25 μg/ml spectinomycin, and 2 μg/ml tetracycline for R. capsulatus; 25 μg/ml streptomycin, 30 μg/ml gentamycin, and 2 μg/ml tetracycline for R. rubrum; 700 μg/ml spectinomycin, and 25 μg/ml tetracycline for R. eutropha. For autotrophic growth selection of transconjugants, petri dishes containing solid-agar Ormerod’s minimal media with 0.4 % w/v malate were incubated in jars that were flushed with a mixture of either 5 % CO2 and 95 % H2 (for R. capsulatus) or 2.5 % CO2, 47.5 % H2, and 50 % air (for R. eutropha).2831

For liquid autotrophic growth, R. capsulatus and R. eutropha transconjugants were

grown in Ormerod’s minimal medium that was bubbled with an appropriate gas mixture. For anaerobic photoheterotrophic growth, R. rubrum transconjugants were placed in


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Ormerod’s sulfur-free minimal medium (sulfate salts replaced with chloride analog) with 0.4 % w/v malate, supplemented with either 500 μM MTA or 1 mM sulfate (positive control).23, 29-31 Molecular biology procedures and plasmids used. Plasmid pCR-RpcbbM contained the

R. palustris cbbM gene in pCR-blunt II-TOPO.23 Plasmid pRPS-MCS3, which contains an R. rubrum cbb promoter and a cognate gene encoding the regulatory protein (CbbR), was used for trans complementation and expression of R. palustris cbbM genes in R.

capsulatus and R. rubrum.8,21,23 Plasmid p90, which contains the R. eutropha cbb promoter, was used for complementation analysis in R. eutropha.28 Plasmids pET11a or pET28a were used for recombinant-gene expression and protein production in E. coli.8,28 Plasmid sequences were verified by Sanger sequencing at the Plant Microbe Genomics Facility (The Ohio State University; OSU) or at the Genomics Shared Resource facility at OSU’s Comprehensive Cancer Center. Specific nucleotide substitutions were introduced in the R. palustris form II RuBisCO gene using a QuickChange Lightning site-directed mutagenesis kit (Agilent), appropriate primers and plasmid pCR-RpcbbM. Random mutagenesis of R. palustris cbbM genes


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encoding mutants M331A, M331L, I165A, or I165T was performed using error-prone PCR amplification with the appropriate pCR-RpcbbM plasmid as template in the presence of 0.1 mM MnCl2 and Taq DNA polymerase (Thermo Fisher Scientific).

R. palustris cbbM genes were amplified using PrimeSTAR GXL DNA Polymerase (Takara) and cloned into plasmid pRPS-MCS3, p90, or pET11a or pET28a (Novagen), as previously described;8 plasmid pRPS-RucbbM with the R. rubrum cbbM gene cloned into pRPS-MCS3 was as previously described21. Plasmids were transformed into E. coli S17 and mobilized into the appropriate RuBisCO-deletion strain of R. capsulatus (strain SB I/II-) or R. eutropha (strain H16LS) or R. rubrum (strain IR) via biparental matings. R.

eutropha was used for suppressor selection.28 Plasmids from original transconjugants and cultures exhibiting growth under the given experimental conditions were re-isolated (mini-prep, Qiagen) and sequence-verified.

Recombinant protein synthesis and purification. For recombinant protein synthesis, the

R. palustris cbbM genes cloned into plasmid pET11a or pET28a, were transformed into E. coli BL21 (DE3). Single-colony isolates were used to inoculate 5 ml of LB media with


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50 ng/μL kanamycin, which was then used to inoculate 0.5 – 1 L of LB media with 30 μg/μl kanamycin in 2.8-liter Fernbach flasks. Cultures were grown to an optical density (at 600 nm) of 0.4-0.8 and protein synthesis was induced by adding IPTG to 1 mM final concentration. Cells were further incubated at 23°C for 12-14 h, harvested, washed and stored as pellets at -20°C. Prior to purification, cells were washed and resuspended in either a 50 mM sodium phosphate buffer, pH 8.0, with 300 mM NaCl and 15 mM imidazole (for Ni-NTA affinity purification) or 50 mM Bicine-NaOH buffer, pH 8.0, with 1 mM DTT, 10 mM MgCl2 and 10 mM NaHCO3 (for non-tagged enzyme purification) and lysed using a French-pressure cell at 1000 psi. Lysates were clarified using high-speed centrifugation (16,000 x g) at 4°C. Hexa-histidine tagged proteins expressed using pET28a were purified using one-step nickel-affinity chromatography (Ni-NTA Agarose, Qiagen) following manufacturer’s protocols except that the imidazole concentrations were 15 mM and 30 mM in sample lysis and wash buffers, respectively, to reduce non-specific protein binding. Non-tagged proteins expressed using pET11a were purified using a two-step procedure with Green Separopore resin (bioWORLD, OH) followed by UnoQ (Bio-Rad) ion-exchange


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chromatography described previously.8 Eluted proteins were concentrated into 50 mM Bicine-NaOH, pH 8.0, 10 mM MgCl2, 1 mM DTT, and 10 mM NaHCO3 using Amicon (Sigma-Millipore) 50 kDa or 100 kDa centrifugation-concentration filters. Protein concentration was determined by Bradford assay (Bio-Rad). Glycerol was added to 20 % final concentration, and aliquots were frozen in liquid nitrogen and stored at -80 °C. For structural studies, target proteins were also purified by a final size exclusion chromatography step using a Superdex S200 column equilibrated with 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 10 mM MgCl2, 20 mM NaHCO3 and concentrated to ~21.5 mg/ml. All of the proteins eluted as hexamers in fractions corresponding to a molecular weight ~300 kDa.

Crystallization, data collection and structure determination. For co-crystallization of RuBisCO enzymes with the transition state analog 2-CABP, a 160 µl aliquot of protein was diluted with 48 µl of CABP (12.9 mM in 100 mM Tris pH 8.5), and 2 µl of storage buffer giving a CABP to protein ratio of 10:1 and a final protein concentration of ~16 mg/ml. The CABP stock used in these studies was prepared as part of a previous study.8


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Hanging drop vapor diffusion crystallization experiments were performed in 96 well plates using commercially available crystallization screens with drop sizes of 210 µl and sample to reservoir solution ratios of 2:1, 1:1, and 1:2. Optimization of lead conditions was in 24well plates with drop sizes of 2-3 µl and protein to reservoir solution ratios of 2:1, 1:1, and 1:2. Trays were incubated at 18°C. Previously described crystallization conditions that were used with R. palustris wild-type RuBisCO, 8 could not be easily reproduced and so de novo crystallization screening was initiated. All the mutant RuBisCO enzymes, i.e., I165T, K192C, M331A, A47V, S59F, A47V/M331A and S59F/M331A, could be crystallized robustly using a reservoir solution containing 0.1-0.2 M sodium sulfate, 0. 1 M Bis-Tris propane, pH 6.5-8.0, and 20-24% PEG3350 with a 1:2 or 1:1 ratio of sample to precipitant. The chimeric R. palustris RuBisCO enzyme in which the last 5 carboxyterminal residues were replaced with 11 residues from the carboxy terminus of the R.

rubrum form II RuBisCO did not crystallize under the same conditions that were used with the other mutant enzymes. Additional crystallization screening was employed, and highquality diffracting crystals were obtained using 0.4 M sodium phosphate (monobasic), 1.6 M potassium phosphate (dibasic), 0.1 M imidazole (pH 8.0), 0.2 M NaCl. All the RuBisCO


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crystals grew to full-size in 3-5 days. Crystals of mutant enzymes were cryoprotected with a quick soak in 23% PEG3350, 5% PEG400, 0.1M Bis-tris propane (pH 8.0), 0.1 M sodium sulfate, 5.8 mM Tris-Cl, pH 8.0, with 87 mM NaCl, 2.9 % glycerol, 2.9 mM MgCl2, and 5.8 mM NaHCO3 prior to flash-freezing in liquid nitrogen. Crystals of the chimeric R.

palustris RuBisCO enzyme were mounted directly from the drop without any additional manipulation, and flash-frozen with liquid nitrogen. Diffraction data were collected in-house at 100 K using a FRE+ rotating anode generator, VariMax confocal optics, HTC detector (Rigaku) and at beamline 24-ID-C at the Advanced Photon Source of Argonne National Lab. Data were processed with XDS32 and structures were solved by molecular replacement using Phaser33 with the wild-type

R. palustris CABP-bound structure (PDB accession code 4LF1) as the search model. One exception to this procedure was the dataset for the untagged A47V/M331A structure, which was processed using the RAPD software pipeline developed at NE-CAT (Argonne National Laboratory). The structures were refined with Phenix.refine34 or Buster-TNT35 using TLS parameters and NCS restraints and the models improved by iterative rounds of manual model building with Coot36 interspersed with refinement cycles. Figures were


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prepared with PyMOL (Schrödinger). Table S1, containing data collection, refinement statistics, and PDB accession codes, was prepared with Phenix.table_one. 34 Calculation of RMSD differences between structures was done with Coot using the secondary structure matching function unless otherwise mentioned.36 All the mutant enzymes crystallized in the P1 space group with either one or two hexameric assemblies in the asymmetric unit (Table S1). The asymmetric unit of the chimeric enzyme contains a single RuBisCO dimer and the hexameric assembly can be generated through symmetry operations.

Determination of enzymatic properties of recombinant RuBisCOs. The kinetic constants kcat (for carboxylation), Kc (Km for CO2), and Ko (Km for O2) were determined from simultaneous radiometric assays performed in vials flushed with either 100% N2 or 100% O2 as outlined previously using NaH14CO3 (Perkin-Elmer).8,28 The KRuBP (Km for RuBP) values were obtained from similar assays performed in sealed vials by varying the concentration of RuBP (Sigma-Aldrich). All kinetic constants were calculated using SigmaPlot (Systat). The substrate specificity (Ω) values were measured with purified


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enzymes (25 to 250 g) and synthesized (1-3H)-RuBP37 in 400 l assays performed in 1.5-ml vials under saturating (1.23 mM) O2 concentrations as previously described.8,28

RESULTS Role of Ile165 and Met331 in CO2 fixation and the isolation of suppressor mutant S59F/M331A. Residues Ile165 and Met331 are near the active-site of the R. palustris form II RuBisCO and equivalent residues in other RuBisCOs are semi-conserved (e.g. Thr173 and Leu335 in spinach RuBisCO). Neither residue is known to participate directly in the RuBP carboxylation mechanism.8 Ile165 is conserved among form II enzymes and is a conserved threonine in form I and valine in form III enzymes. Similarly, Met331 is conserved among most RuBisCOs, otherwise it is replaced by leucine. Replacement of Ile165 with an alanine or threonine, and Met331 with an alanine or leucine all resulted in enzymes that could not support CO2-dependent growth of the R. capsulatus RuBisCOdeletion strain; only the conservative I165V mutant retained functional competency in vivo (Figures 1 and S1A, Table 2).8 Suppressor-selection in R. capsulatus identified an A47V substitution that resulted in the ability of the M331A negative-mutant to complement and


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support CO2–dependent growth.8 However, no second-site suppressors were obtained with the other negative mutants (i.e., I165A, I165T, and M331L), although a true revertant for I165T (i.e., T165I) and a pseudo revertant for I165A (i.e., A165V) were identified. 8 It was surmised that additional suppressors could be obtained using the RuBisCOdeletion strain of the fast-growing aerobic chemoautotroph, R. eutropha,28 as the host for selection after random mutagenesis. Functional selection in R. eutropha allowed for the identification of a new suppressor, S59F, for mutant M331A. To compare with a previously identified suppressor (A47V/M331A),8 the S59F/M331A suppressor-mutant gene was transferred into R. capsulatus strain SB I/II- and the CO2-dependent growth complementation phenotype was confirmed (Table 2, Figure 1). To better understand the mechanism of suppression, an S59F single-mutant was constructed by site-directed mutagenesis of the wild-type gene. Both S59F and S59F/M331A mutants were able to complement R. capsulatus strain SB I/II- to CO2-dependent growth (Table 2, and Figure 1). Mutant enzymes were purified as recombinant proteins after expression in E. coli and the catalytic properties were determined (Table 3). As with the previously identified A47V/M331A suppressor mutant enzyme,8 the Km values for CO2 and O2 were both


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substantially higher and the substrate specificity factor, , was lower for the S59F/M331A mutant enzyme relative to wild-type values. In addition, the S59F/M331A double-mutant enzyme had a significantly lower kcat value relative to the wild type. Like the A47V substitution, the S59F substitution appears to function by lowering the KRuBP value of the M331A negative mutant enzyme (Table 3).

Figure 1. Growth studies with Met331 and suppressor RuBisCO mutants. Anaerobic photoautotrophic growth of R. capsulatus strain SBI/II- complemented with an empty plasmid (i.e., no RuBisCO-encoding gene present; pRPS-MCS3) or with pRPS-MCS3 plasmids expressing the various R. palustris (Rp) mutant form II RuBisCO genes in liquid cultures bubbled with 5% CO2/95% H2 gas. Error bars are standard deviations (n = 3)


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calculated from three independent cultures for each strain. Growth was followed by measuring the optical densities of liquid cultures at 660nm (O.D.660 nm).

Table 2. Growth phenotypes conferred by wild type and mutant R. palustris form II RuBisCOs.

In vivo test for:

CO2-Dependent

MTA Metabolism

Growth Reporter Strain: Carbon/Sulfur

R. capsulatus SBI/IICO2 / SO42-

R. rubrum IR Malate / MTA

Malate / SO42-

Source: Rubisco gene

Lag (h) Doubling (h)# Lag (h)

Doubling

Lag (h) Doubling (h)#

(h)# None

N.G.*

N.G.*

228 ± 14

112 ± 4

111 ± 3

23 ± 3†

Rr wild-type

62 ± 4

15 ± 4†

140 ± 3

38 ± 3†

95 ± 3

21 ± 3†

Rp wild-type

86 ± 6

20 ± 5†

116 ± 6

35 ± 3†

105 ± 2

22 ± 2†

Rp M331A

N.G.*

N.G.*

120 ± 3

35 ± 3†

87 ± 5

27 ± 4†

Rp A47V

82 ± 5

21 ± 4†

128 ± 3

34 ± 3†

100 ± 3

24 ± 3†

Rp A47V, M331A 254 ± 17

65 ± 12

123 ± 4

29 ± 3†

64 ± 6

27 ± 5†

Rp S59F

84 ± 4

18 ± 4†

116 ± 5

36 ± 5†

93 ± 3

22 ± 3†

Rp S59F, M331A 245 ± 9

82 ± 11

113 ± 5

35 ± 5†

70 ± 3

24 ± 3†

Rp I165V

81 ± 4

21 ± 4†

210 ± 3

30 ± 3†

85 ± 5

27 ± 4†

Rp I165T

N.G.*

N.G.*

180 ± 3

55 ± 3

85 ± 3

18 ± 3†

Rp E49A

N.G.*

N.G.*

148 ± 9

33 ± 8†

94 ± 9

28 ± 9†

Rp E49G

N.G.*

N.G.*

162 ± 8

39 ± 7†

105 ± 8

26 ± 8†

Rp T54A

N.G.*

N.G.*

154 ± 7

38 ± 7†

101 ± 6

27 ± 5†

Rp N112A

N.G.*

N.G.*

149 ± 7

34 ± 7†

81 ± 9

19 ± 9†


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Rp N112K

N.G.*

N.G.*

196 ± 10

49 ± 8†

89 ± 10

20 ± 8†

Rp N112Q

N.G.*

N.G.*

140 ± 7

30 ± 6†

89 ± 8

23 ± 8†

Rp N112S

N.G.*

N.G.*

152 ± 15

129 ± 16

117 ± 10

30 ± 8†

Rp K167G

N.G.*

N.G.*

218 ± 10

89 ± 21

94 ± 4

31 ± 8†

Rp K167A

N.G.*

N.G.*

305 ± 6

90 ± 11

99 ± 9

25 ± 8†

Rp K167R

N.G.*

N.G.*

296 ± 6

93 ± 10

101 ± 10

29 ± 9 †

Rp K169A

N.G.*

N.G.*

215 ± 20

62 ± 6

97 ± 10

27 ± 9†

Rp K192A

N.G.*

N.G.*

294 ± 6

86 ± 7

96 ± 7

25 ± 7†

Rp K192C

N.G.*

N.G.*

191 ± 8

93 ± 14

84 ± 4

23 ± 3†

Rp K192R

N.G.*

N.G.*

283 ± 7

91 ± 7

83 ± 10

19 ± 8†

Rp K330A

N.G.*

N.G.*

133 ± 10

26 ± 8†

89 ± 6

18 ± 5†

# Growth data were fit by weighted nonlinear regression to a sigmoidal-logistic model23 to determine the lag time (h) required for the culture to reach 50% of maximum density and the doubling time (h) for the culture optical density measured at 660 nm. Reported error is the 95% confidence interval of the fit parameters when the regression model is weighted using the growth data standard deviation for n = 3 independent growth experiments. * N.G. No Growth observed. † Doubling time statistically the same as that observed for when the same strain expresses the R. palustris wild type RuBisCO enzyme under the same growth conditions (P > 0.005, df = 4).

Table 3. Kinetic properties of recombinant R. palustris form II RuBisCO enzymes. Enzyme

Ωa (VcKo/VoKc )

Wild type

10±1

kcat a

Kc a

Ko a

(sec-1)

(M)

(M)

3.8±0.3

102±9

309±2

Ko/Kc b KRuBP a (M) 3.0

11±4

1


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Biochemistry

M331A*

7

0.2

338

3914

11.6

127

A47V*

9

0.7

97

91

0.9

7

A47V/M331A*

5

0.2

425

778

1.8

22

2.8±0.5

75±6

141±1

1.9

9±1

0.62±0.0

698±3

803±8

1.2

16±2

4

8

1

S59F S59F/M331A

11±1 7±1

4

*Values obtained from a previous study8 and normalized against wild-type values presented here a Values are the means ± standard deviation (n-1) of at least three separate enzyme preparations b Calculated from measured Kc and Ko values Abbreviations used: Vc - Vmax for carboxylation; Vo - Vmax for oxygenation; Kc – Km for CO2; Ko – Km for O2; KRuBP – Km for RuBP

Movement of the N-terminal domain towards the active-site comprises a key step in the later stages of RuBisCO’s catalytic mechanism involving CO2 addition to the 2,3-enediol intermediate.38 Interestingly, sequence alignments indicated that RLP residues that diverge from conserved RuBisCO active-site residues are primarily located in this region.9 Residues Ala47, Ser59 and Met331 are located in an interface formed by residues in the C-terminal domain of one large subunit and N-terminal domain of the adjacent large subunit (Figure 2). Because substitutions Ala47 and Ser59 in the M331A mutant enzyme


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overcome the negative growth phenotype and enzymatic properties by altering the KRuBP values (Table 3), further studies aimed to determine whether Met331 or its suppressors properly structured the RuBisCO active site for enolization of RuBP. Towards this end, the ability of these RuBisCO mutants to support MTA metabolism in R. rubrum was used as an indirect indicator of their potential ability to catalyze RuBP enolization. Thus, all the mutant genes were expressed in trans into R. rubrum strain IR21,23 and tested for their ability to support RubisCO-dependent growth with MTA as sole sulfur source. Growth rates for strain IR expressing the mutant genes encoding M331A, A47V, S59F, A47V/M331A, A47V/S59F, I165T, were statistically the same as the wild-type enzyme (P > 0.05, df = 4) and mutant I165V conferred intermediate growth (Table 2), indicating that amino-acid substitutions at these residues likely retain the ability to catalyze the initial steps in the RuBisCO catalytic mechanism. In conclusion, residues Met331 and Ile165 and their interactions appear to be specific and critical for CO2 addition onto the 2,3enediol(ate) intermediate derived from RuBP.


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Biochemistry

Figure 2. Comparison of inter-subunit interfaces between the N-terminal and C-terminal domains of a dimeric pair of large subunits that form a bipartite RuBisCO active site in R.

rubrum (A) and R. palustris (B) form II RuBisCOs. Ligand binding (indicated by the presence of RuBP or CABP and magnesium ion) results in the formation of a “closed” conformation mediated by the movement of loop 6 and another loop region between helix B and -sheet C of the N-terminal domain in the neighboring large subunit (magenta). (A) Structural superposition of the interface in the “open” unactivated (PDB ID 5RUB; light brown) and “closed” activated RuBP-bound (PDB ID 9 RUB; teal) R. rubrum form II RuBisCO. Arrowheads 1 and 2 mark the boundaries of the disordered loop 6 (residues Thr324 to Ser335), and arrowheads 3 and 4 mark the boundaries of the


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disordered N-terminal domain loop (residues Asn54 to Thr64) in the open structure. (B) Structural superposition of “closed” CABP-bound (PDB ID 4LF1l dark gray) and sulfatebound (PDB ID 4LF2; light gray) structures of R. palustris form II RuBisCO. All pertinent residues are shown in stick configuration and are labeled with residues from the Cterminal domain of a large subunit of the dimeric pair indicated with asterisks (*).

RuBisCO residues required for RuBP enolization and CO2 fixation. Conserved catalytic residues Glu49, Thr54, Asn112, Lys167, Lys169, Lys192 and Lys330 (Glu60, Thr65, Asn123, Lys175, Lys177, Lys201 and Lys334 in spinach RuBisCO) have all been implicated in various steps of RuBisCO’s catalytic mechanism based on previous biochemical studies.6,18,39,40-60 To delineate the role of these residues in RuBP enolization versus CO2 fixation steps of the catalytic mechanism, we constructed mutant enzymes E49A, E49G, T54A, N112A, N112K, N112Q, N112S, K167A, K167G, K167R, K169A, K192A, K192C, K192R, and K330A. The identities of amino-acid substitutions introduced were largely prompted by previous structure-function studies with the analogous R.

rubrum form II RuBisCO

18,39,40-60

None of these R. palustris form II RuBisCO-mutant


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Biochemistry

enzymes restored CO2-dependent growth of R. capsulatus strain SB I/II- (Table 2 and Figure S1), demonstrating compromised RuBP carboxylase function. When expressed in

R. rubrum strain IR, photoheterotrophic growth on MTA remained poor for mutants N112S, K167A, K167G, K192A, K192C and K192R, identical to that seen for the empty plasmid control (P > 0.05, df = 4) (Table 2). This indicated that both the enolization and CO2-addition steps of the catalytic mechanism were compromised in these mutant enzymes. Mutants N112K and K169A supported intermediate growth between that of the wild-type enzyme and empty plasmid control. Mutants E49A, E49G, T54A, N112A, N112Q, and K330A conferred wild-type rates of MTA-dependent growth (P > 0.05, df = 4) (Table 2). These results confirmed that the identities of Lys167 and carbamylated Lys192 are absolutely critical for stabilizing the RuBP 2,3-ene-diol(ate) intermediate in addition to their possible roles in the later stages of the mechanism, whereas Glu49, Thr54, Lys169, and Lys330 are primarily responsible for coordinating the incoming CO2 molecule during carboxylation.6,18,39,42 Structural features of mutant and chimeric R. palustris RuBisCO enzymes. The availability of more than 100 structures of divergent RuBisCOs and RLPs in the protein


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data bank has facilitated the identification of several structural features and conformational changes that are critical for the catalytic mechanism. As indicated earlier, one such structural transition that is essential for carboxylation of the 2,3-ene-diol(ate) intermediate is the closure of the active-site loop six in one of the large subunits and the concomitant movement of an N-terminal loop of residues (between B and C) from the neighboring large subunit of the dimeric pair to stabilize the catalytic loop six conformation.38,42,61,62 This is evident from comparison of the N-terminal domain/catalytic loop six inter-subunit interface in the “open” unliganded and intermediate liganded (activated, with RuBP) structures of R. rubrum form II RuBisCO (Figure 2A). The conformational flexibility of the region from Gly52 to Thr64 in the N-terminal domain of one subunit and Thr324 to Ser334 in the loop six of the neighboring subunit in R. rubrum RuBisCO (Figure 2A), or equivalent regions in other RuBisCOs, are critical for gaseous substrate binding after the enolization of RuBP has been completed.61,62 The equivalent residues in both the “closed” liganded R. palustris RuBisCO structures (activated, with CABP or sulfate ions) are all ordered (Figure 2B). The conformations of some residues at the interface are noticeably different between the sulfate bound and CABP-bound


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Biochemistry

structures (Figure 2B), which is likely a consequence of the packing of RuBisCO subunits within the crystal lattice. A completely “closed” conformation, which is usually represented by tightly bound ligands in the active site that are completely shielded from the solvent, is a prerequisite for catalysis.38,61,62

Structures of M331A- and suppressor-mutant enzymes highlight the importance of Nterminal residue interactions with CABP. Inter-subunit electrostatic interactions between the N-terminal domain active-site residues Glu49, Thr54, Asn55 and Asn112 (Glu60, Thr65, Trp66 and Asn123 in spinach RuBisCO) with CABP and active-site residues Lys330 and Lys169 (Lys334 and Lys177 in spinach RuBisCO), as well as van der Waals interactions of Met331 and Lys330 with Asn112, Gly115, Met116, and Gly117, appear to help stabilize the “closed” conformation of catalytic loop six.62 This is reinforced by additional points of contacts between the N-terminal loop residues and the C-terminal domain of the neighboring large subunit of the R. palustris enzyme (Figures 2 and 3). The active site of the CABP-bound M331A-mutant structure does not show significant differences in the positions of these N-terminal domain active-site residues directly


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Page 28 of 66

interacting with the loop six region. However, the conformation of several residues (Glu57, Asp63, and Arg66) in the loop between B and C in the N-terminal domain appear to be perturbed (Figure 3A). Given the kcat values of 0.2 s-1 for M331A versus 4.9 s-1 for the wildtype enzyme (Table 3), it is likely that closure of loop six and stabilization by the N-terminal domain is a rare event in the mutant M331A enzyme compared to the wild type. Thus, it is not surprising that the suppressor substitutions A47V and S59F that were obtained for M331A both reside in the same N-terminal region. Both substitutions result in the replacement of shorter side chains with bulkier hydrophobic groups, thereby stabilizing the respective local hydrophobic environments and the entire N-terminal domain in the “closed” conformation (Figure 3, B and C). Structural analysis of mutant enzymes reinforces the importance of non-catalytic residue Met331 and the surrounding N-terminal domain in structuring the RuBisCO active site for proper RuBP carboxylase function.


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Biochemistry

Figure 3. Comparison of the inter-subunit interfaces in R. palustris form II wild-type and mutant RuBisCO X-ray crystal structures. (A) Superposition of wild-type (PDB ID 4LF1; dark gray) and M331A-mutant (PDB ID 5HAO; light orange) RuBisCOs, showing subtle differences in the positions of the side chains of N-terminal residues Glu57, Asp63 and Arg66. (B) Alignment of M331A-mutant RuBisCO structure (PDB ID 5HAO; light orange) with the structures of A47V-single (PDB ID 5HJX; light blue) and A47V/M331A-double (PDB ID 5HQL; dark blue) mutant RuBisCOs. An A47V substitution introduces new van der Waals interactions with the side chains of Val72 and Ile84 (dotted lines). (C) Alignment of M331A-mutant RuBisCO structure (PDB ID 5HAO; light orange) with the structures of S59F-single (PDB ID 5HAN; light green) and S59F/M331A-double (PDB ID 5HAT; dark green) mutant enzymes. Black dotted lines indicate polar (involving side chain oxygen of Ser59 in mutant-M331A structure; light orange) and non-ideal interactions (involving


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phenyl-ring carbons in S59A-single and S59A/M331A-double mutant structures; green) with the carbonyl oxygen atoms of Phe398, Gly399, and His400. Hydrophobic interactions between Phe59, Val58 and Val401 in the structures of S59F-single and S59F/M331Adouble mutant enzymes are also indicated with black lines. The superimposed structures in panel C have been rotated along the z-axis relative to the orientations shown in panels A and B to better represent the interactions involving Ser/Phe59.

Salient features of the I165T-mutant enzyme structure and engineering a form I-like active site. The structure of wild-type R. palustris form II RuBisCO indicates that the side chain of Ile165 is also in a hydrophobic region. Steric interactions in this region likely play a role in correctly positioning the carbamylated Lys192 and Ser369 catalytic residues (Lys201 and Ser379 in spinach RuBisCO) (Figure 4A). Carbamylated Lys192 is essential for magnesium coordination, RuBP proton abstraction, and stabilizing the RuBP 2,3-enediol(ate) intermediate.6,55-57,61,62 Ser369 is involved in CO2/O2 specificity and later aspects of the carboxylation mechanism.45,60,61 When Ile165 is substituted with threonine, the side chain hydroxyl moves within hydrogen-bonding distance of oxygen O2 of CABP and a


30

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Biochemistry

carbamyl oxygen of Lys192, much like the arrangement in the CABP-bound spinach enzyme (Figure 4, B and C). Even though this appears to stabilize the position of Lys192, re-orientation of Ser369 also results from the loss of a hydrogen bond between the hydroxyl group of Ser369 and oxygen O4 of CABP in exchange for a new hydrogen bond with the hydroxyl group of Thr392 (Figure 4B). These altered interactions likely result in an active site that is still able to perform enolization of RuBP and a putative substrate in MTA metabolism, but cannot efficiently fix CO2 due to the loss of CABP’s interaction with Ser369, which is essential for later aspects of the carboxylation mechanism post RuBP enolization.45,63,64 These structural changes induced by I165T thus explain why this mutant enzyme complements for MTA-dependent growth but was unable to process enough RuBP molecules via the carboxylation mechanism to sustain CO2-dependent growth.


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Figure 4. Comparison of active sites in wild-type and mutant RuBisCOs. View of the CABP-bound active sites of (A) the wild-type R. palustris form II enzyme (PDB ID 4LF1; dark gray), (B) form I spinach RuBisCO (PDB ID 8RUC; yellow), (C) I165T-mutant (PDB ID 5HJY; cyan), and (D) K192C-mutant (PDB ID 5KOZ; pink) R. palustris form II RuBisCOs. Active site residues and the CABP molecule are shown in stick representation while the magnesium ion is represented by a sphere. CABP and magnesium ions are labeled in panel A. In contrast with the other structures (A-C), the active site of the K192Cmutant enzyme (D) contains a water molecule (indicated by a red sphere) and a


32

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Biochemistry

carbonate ion; two of the oxygens from the carbonate ion occupy similar positions as the OQ1 and OQ2 oxygens of the carbamylated side chain of residue Lys192 in the wild-type structure (A). Amino acid residues are labelled with those from the N-terminal domain of a second subunit comprising the active site indicated with an asterisk (*). Unique hydrogen bond interactions are indicated in each structure with dotted black lines.

The X-ray crystal structure of I165T mutant enzyme provided a structure-based rationale for introducing T392Q and A393F as potential second-site suppressors. It was surmised that this would mimic the structural arrangement in a form I RuBisCO around the residue Thr165, as in the structure of spinach RuBisCO (Figure 4, A-C). However, neither substitution, when present alone or in combination, could suppress the negative phenotype of R. palustris mutant I165T upon testing for the capacity to support CO2dependent growth in R. capsulatus SB I/II- (Figure S2). These findings likely indicate that other structural differences between the form I and form II RuBisCO enzymes contribute to positioning Ile165 (in R. palustris form II) or the equivalent Thr173 (in spinach form I RuBisCO) in the active site for optimal catalysis.


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Page 34 of 66

Structural analysis of active-site interactions involving the catalytic residue Lys192. Unlike Ile165, which resides in the second sphere of coordination around the active site, residue Lys192 has a direct role in catalysis. Activation of the enzyme via -amino carbamylation of this catalytic lysine is necessary for both the RuBisCO carboxylation mechanism55 and the enolization mechanism of at least one RLP class, i.e., the Bacillus family YkrW/MtnW RLP.10-12 A previous study showed that substitution of equivalent Lys191 in R. rubrum RuBisCO with cysteine resulted in a mutant enzyme devoid of CO2 fixation activity but still retained tight-binding with CABP, a property characteristic of activated RuBisCOs.56,57 This was the rationale for the structural analysis of R. palustris K192C mutant RuBisCO. The mutant enzyme was indeed amenable to crystallization with CABP and the structure showed that the absence of an -amino group on this residue resulted in the loss of coordination with CABP via the magnesium ion (Figure 4D). However, a carbonate ion is bound in the active site of the K192C-mutant enzyme with the two oxygen atoms occupying the positions of the two carbamyl oxygens (OQ1 and OQ2) in the wild-type structure. The hydrogen bond interactions between the carbonate


34

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Biochemistry

ion and the magnesium ion, CABP, and the side-chain oxygen atoms of Asp194 and Gly195 are similar to those formed by the lysyl carbamate ion in the wild-type structure (Figure 4A), explaining why the mutant enzyme is able to bind tightly to CABP despite its complete loss of activity. Aside from these interactions, the K192C-mutant enzyme structure has additional hydrogen bond interactions that are not seen in the wild-type structure. These include the interaction of the carbonate ion with the main-chain nitrogen of Glu195, and with the side chains of Thr392 and His322 via a water molecule (Figure 4, A and D). The loss of direct interactions involving the carbamylated Lys192 and other altered interactions account for the inability of this mutant enzyme to complement for either CO2-dependent or MTA-dependent growth (Table 2). It can thus be concluded that Lys192 activation via carbamylation likely plays a role in RuBP enolization, similar to what has been shown for the equivalent residue in the structure of YkrW/MtnW RLP.10-12,56

Structure of chimeric form II RubisCO. A previously described chimeric R. palustris form II RuBisCO, which has a substantially enhanced kcat than the wild-type enzyme,8 has the last five residues in the C-terminus swapped with the 11 carboxy-terminal residues of the


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R. rubrum form II RuBisCO.8 X-ray crystal structure of the chimeric RuBisCO superimposes well with the wild-type structure (rmsd of 0.48 Å for the superposition of 453 cα atoms) with no discernible differences in the active site region or any of the secondary-structural elements (Figure S3). Electron density was observable for only the first five residues in the chimeric carboxy terminus corresponding to residues Gly457Thr461 of the R. rubrum sequence G(457)VEDTRSALPA(467). The additional residues represent a mobile region that likely affects catalysis in ways not discernible from a structural snapshot. The chimeric enzyme did not crystallize under the same conditions as the other mutant RuBisCOs studied in this project and optimal conditions for growing diffraction quality crystals had to be determined from crystallization screening. The new crystal form determined for the chimeric enzyme has only a single RuBisCO dimer in the asymmetric unit, however, the hexameric assembly seen for the other mutants crystallized in this study can be generated through symmetry operations. Hexameric assembly of the chimeric enzyme was confirmed by size exclusion chromatography. It is likely that the chimeric part of the carboxy terminus plays a critical role during


36

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Biochemistry

conformational-state changes of the enzyme during catalysis, which is difficult to capture via X-ray crystallography.

DISCUSSION Mutational studies with representative RuBisCOs from plants (form I), bacteria (forms I and II), and archaea (form III) have identified residues that function in inter- and intrasubunit interactions, active-site structuring, substrate binding and specificity, and catalysis.7,14,65 Structure-function studies with the R. palustris form II RuBisCO presented here and in a previous study8 confirmed that the semi-conserved residues Ile165 and Met331 (Thr173 and Leu335 in spinach RuBisCO) near the enzyme’s active site are indeed critical for RuBisCO function, as shown previously with R. rubrum,

Chlamydomonas, Synechococcus and tobacco RuBisCOs.65-69 The present studies also provide additional insights regarding the importance of N-terminal domain movement relative to the active site in the C-terminal domain of the neighboring subunit for catalysis, as suggested previously.6,38,61,62 When combined with the analysis of active-site residues, including those at this inter-subunit interface, we now have a better understanding of the


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Page 38 of 66

structural interactions that are critical for different steps in the enzyme’s catalytic mechanism.

Importance of the residue interactions at the N-terminal domain/catalytic loop 6 interface. M331A or M331L substitutions in the R. palustris form II RuBisCO and an equivalent M330L substitution in the R. rubrum form II RuBisCO all resulted in similar detrimental changes to the enzyme’s kcat, CO2/O2 specificity, and KRuBP values (Table 3).8,67 Even though the increased KRuBP values could suggest a possible role for this residue in RuBP binding and enolization, detailed characterization of the reciprocal L332M substitution in cyanobacterial form I RuBisCO and an L335V substitution in tobacco RuBisCO led to the conclusion that this residue is only involved in the later steps of the carboxylation mechanism.65,69 Therefore, it is not surprising that amino-acid substitutions in this region of the R. palustris form II RuBisCO have very little impact on the ability to support MTA-dependent growth, indicative of enolization functionality, but are detrimental to RuBP carboxylation (Table 2). This is further substantiated by the inability of mutants E49A, E49G, T54A, N112A, N112Q, and K330A to support CO2-


38

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dependent growth despite retaining competency to support wild-type levels of MTAdependent growth of R. rubrum strain IR (Table 2). The poor kinetic properties conferred by the M331A substitution (Table 3) and the complete loss of carboxylation activity caused by other amino-acid substitutions in this region likely result from the disruption of the inter-subunit interface that optimally positions catalytic residues Glu49, Thr54, Asn55, Asn112 and Lys330 for interactions with the 2,3-enediol(ate) intermediate during catalysis. The inability of the R. palustris N112S and N112K mutants to complement R. capsulatus strain SB I/II- for CO2-dependent growth and their severely compromised abilities to support MTA-dependent growth of R. rubrum strain IR (Table 2) are attributable in part to the ~95% loss of RuBP enolization activity, as reported for the equivalent R. rubrum mutant enzymes.47 However, mutants enzymes N112Q and N112A, which are unable to complement R. capsulatus strain SB I/II- for CO2-dependent growth, fully complement MTA-dependent growth of R. rubrum strain IR (Table 2). This is corroborated by the observation that the N111Q mutant of R. rubrum RuBisCO retained 30-40% of the wildtype level of RuBP enolization activity, and the KRuBP value measured with an N123G


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mutant of Chlamydomonas form I RuBisCO was comparable to the wild-type value. These observations confirm that this Asparagine residue does not have a direct catalytic role in enolization of RuBP and rather indicate a structural role in in this region of the active site for both enolization and subsequent carboxylation steps.47,48 Similarly, amino-acid substitutions at the residue equivalent to R. palustris Lys169 in the R. rubrum enzyme (Lys168) retained 2-3% enolization activity relative to the wild type while only retaining < 0.01 % overall carboxylase activity, leading to the conclusion that this residue is not directly involved in the enolization of RuBP. Rather, like Asp112, Lys169 appears to play a structural role by forming a salt bridge with the side chain of Glu48 from the N-terminal domain of the neighboring subunit.18,42 This is consistent with the observation that mutant K169A is defective in CO2-fixation, but can support, albeit poorly, MTA-dependent growth, indicative of residual enolization activity. Suppressor mutant A47V is located within the B helix two residues away from Glu49, which is essential for inter-subunit stabilization via the formation of a salt bridge with Lys169 from the adjacent subunit, and for coordinating with the catalytic Lys330 during 2,3-ene-diol(ate) carboxylation.42,61 The A47V mutant structure shows that the bulkier


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hydrophobic valine side chain is now in close proximity with Val72 and Ile84 side chains within the βC and βD strands. The resulting van der Waals interactions between these residues stabilize the local environment between the βC/βD strands and the B helix face opposite Glu49 (Figure 3). Unlike A47V, the S59F substitution occurs at the inter-subunit interface and likely has a direct impact on the stability of this interface (Figure 3, B and C). The hydroxyl group of Ser59 is 3.3 Å away from the main-chain oxygen of Phe398, possibly forming a hydrogen bond in the “closed” conformation of the enzyme, which would be disrupted by the substitution. In addition, this also results in the placement of a bulkier phenylalanyl side chain within close proximity to other hydrophobic residues in the region, while also making some non-ideal contacts with the carbonyl oxygens of Phe398, Gly399, and His400 (Figure 3C). Thus, the S59F substitution appears to function by balancing these opposing steric interactions at the inter-subunit interface when forming a “closed” conformation in both the S59F and S59F/M331A mutant enzymes. These subtle changes in the positions of N-terminal domain residues in mutant M331A and its suppressors all result in substantial differences in the Km values for gaseous substrates CO2 and/or O2 compared to the wild-type enzyme (Table 3).


Rather, the altered

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interactions imposed by the suppressors appear to be primarily functioning by restoring the KRuBP of the M331A enzyme to near wild-type values. Detrimental effects of N-terminal domain amino acid substitutions on the CO2/O2 specificity, but not KRuBP, have a precedence. Directed evolution studies with the R. rubrum RuBisCO identified amino-acid substitutions in this region at residues His44 and Asp117 (His45 and Asp118 in R.

palustris form II RuBisCO).70 His44 of the R. rubrum enzyme (conserved in all form II RuBisCOs) resides in the αB helix opposite Ala46 and forms a hydrogen bond with the side chain of Asp117 (Ala47 and Asp118 in R. palustris form II RuBisCO). Disruption of these hydrogen-bond interactions resulted in destabilization of this same N-terminal domain/catalytic loop six inter-subunit interface, leading to a significant change to the enzyme’s specificity for CO2 versus O2, but not to the KRuBP values.70

Importance of interactions involving Ile165 and active-site residues that are absolutely essential for both RuBP enolization and CO2 addition. It has not been possible to isolate a second-site suppressor for either mutant I165A or I165T using random mutagenesis and biological selection or by introducing residue substitutions deduced from the I165T


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mutant-enzyme structure. Notably, an equivalent substitution in the R. rubrum form II RuBisCO (I164T) and a reciprocal substitution of T173I in the Chlamydomonas form I RuBisCO (similar to spinach RuBisCO) both result in near complete loss of carboxylase activity.66,68 The re-orientation of Ser369 in the R. palustris I165T-mutant RuBisCO structure (Figure 4C) likely explains the detrimental changes observed in the enzyme’s RuBP carboxylase catalytic properties.8 Catalytic residue Lys192 (Lys201 in spinach RuBisCO) is responsible for both coordinating the magnesium ion necessary for catalysis and serving as the essential base for C3 proton abstraction of RuBP.6,18,61 Substitutions at this lysine residue render the enzyme devoid of all catalytic activity, including enolization of RuBP and carboxylation of the 2,3-ene-diol(ate) intermediate, unless a chemical modification mimicking the carbamylated lysine is reintroduced.55-57 The presence of a carbonate ion in the active site of the K192C-mutant structure in place of the carbamyl group usually present in the wild-type enzyme (Figure 4D) points to the requirement of electronegative groups to stabilize the cofactor magnesium ion, which is central to the enolization mechanism. However, rearrangement of multiple interactions around the active site explains the


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inability of this mutant to participate in key catalytic events that may be required for either function. Structural and computational analyses indicate that after C3 proton abstraction by Lys192, it is transferred to the catalytic residue Lys167 (Lys175 in spinach RuBisCO), which is positioned to donate it back to the aci-carboxylate form of the upper 3PGA to complete the last step of the carboxylation mechanism.18,54 A K166G substitution of the equivalent R. rubrum RuBisCO residue rendered the enzyme devoid of all enolization activity and aci-acid protonation activity. Yet, the enzyme retained activity for hydrolysis and carbon-carbon cleavage of the carboxylated reaction intermediate, 2-carboxy-3ketoarabinitol 1,5-bisphosphate, at 4% of the wild-type level, indicating that this residue was specifically involved only in the initial enolization and the terminal aci-acid protonation steps.53,54 Equivalent substitutions of both Lys192 and Lys167 in the B. subtilis RLP to isoleucine, glutamic acid, or alanine all resulted in a complete loss of enolization activity as well.12 The inability of Lys167 and Lys192 mutants of R. palustris form II RuBisCO to support either CO2-dependent or MTA-dependent growth (Table 2) is consistent with the possible role for these residues in the enolization of RuBP.18


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Structural features of chimeric R. palustris form II RuBisCO. The X-ray crystal structure of chimeric R. palustris form II RuBisCO with the R. rubrum form II enzyme’s carboxy terminus provides a snapshot of the enzyme that likely resembles the conformational state after CO2 addition to the 2,3-ene diol(ate). The folded conformation of the carboxy terminus over loop six in form I RuBisCOs is linked to the regulation of active site opening and closure rates and thus the catalytic rates.62 However, the presence of an extended conformation of the carboxy terminus and its placement away from the active site in R.

rubrum and R. palustris wild-type, and R. palustris chimeric form II enzymes (Figure S3) is indicative of steric constraints that may prevent the placement of the carboxy terminus over loop six. Because no significant differences are discernible from the alignment of active sites in the wild-type and chimeric enzymes, the molecular basis for the higher kcat observed with the chimeric enzyme8 could not be delineated. However, it is intriguing that the crystal form of the chimeric enzyme contained only dimeric units in place of a hexameric unit. Despite this observation and the need for employment of different crystallization conditions for the chimeric enzyme, size-exclusion chromatography confirmed that the recombinant enzyme indeed assembled as a hexamer. The


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observation of dimeric units with the chimeric enzyme could also be an artifact arising from the use of a higher salt concentration for crystallizing this particular protein. Truncations of the longer native carboxy terminus in the R. rubrum RuBisCO led to the formation of octameric holoenzyme molecules, which was attributed to the exposure of a hydrophobic patch of residues.71 In an earlier study, a homologous form II RuBisCO from

Rhodobacter sphaeroides was reported to form higher order oligomers in a pH-dependent manner.72 Based on these observations, it is possible that transient changes in the structural states of the holoenzymes may impact the catalytic performance in subtle ways not discernible from a structural snapshot, possibly explaining the differences in properties of the wild-type and chimeric R. palustris form II RuBisCO enzymes.

Summary. This study combines structural and functional analysis of mutant R. palustris form II RuBisCO enzymes to better define the roles of different structural regions in the enzymatic mechanism. X-ray crystal structures of the mutant enzymes described here provide the first structural snapshots of functionally altered form II RuBisCO enzymes, with the active-site residue conformations closely representing the enzyme bound to the


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6-carbon intermediate (i.e., after CO2 addition). It is evident from these studies that the movement of an N-terminal region in one subunit relative to the active site in the Cterminal domain of the neighboring large subunit plays an important role in the steps that follow the enolization of substrate RuBP. Moreover, the various active-site mutants generated as part of this study will help define RuBisCO’s specific function in the MTAdependent sulfur-salvage pathway. The carboxylation negative mutant enzymes identified as part of this study will serve as useful templates for suppressor-selection studies to identify additional complementing structural interactions.

ASSOCIATED CONTENT Supporting Information. Liquid growth complementation phenotypes of R. capsulatus strain SB I/II- complemented with wild-type and mutants of R. palustris form II RuBisCO genes (Figure S1), CO2-dependent growth complementation phenotypes of R. palustris form II RuBisCO mutants in R. capsulatus SB I/II- (Figure S2), Superposition of the structures of R. palustris form II wild-type and chimeric enzymes (Figure S3), and X-ray crystallography: data collection and refinement statistics (Table S1).


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Accession codes. PDB ids for structural data: 5HJY, I165T; 5KOZ, K192C; 5HQM, Chimera; 5HAO, M331A; 5HJX, A47V; 5HK4, A47V/M331A; 5HAN, S59F; 5HAT, S59F/M331A.

AUTHOR INFORMATION

Corresponding Author * Department of Microbiology, The Ohio State University, Columbus OH 43210, USA. Email: [email protected]. Phone: +1-614-292-4297. Fax: +1-614-292-6337.

Present Address #Air

Force Research Laboratory, Wright-Patterson AFB, Ohio, USA.

Author Contributions S.S., J.A.N., M.A.A. and F.R.T. designed the experiments. S.S., J.A.N., S.N.H., J.A.W. and K.M.B. performed growth complementation analyses. V.A.V. isolated the suppressor mutant. S.S., M.A.A., and A.S. performed the enzyme and crystallographic studies. J.A.N., S.S., and M.A.A. did compilation and statistical analysis of growth, enzymatic, and


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crystallographic data. F.R.T. supervised the research. S.S. and J.A.N. co-wrote the manuscript with contributions and edits from the other authors. All authors have given approval to the final version of the manuscript.

Funding Sources We thank the Center for Applied Plant Sciences (CAPS) at The Ohio State University for partial support to S.S and J.A.N. The UCLA-DOE Institute is supported by the U.S. Department of Energy, Office of Biological and Environmental Research (BER) program under Award Number DE-FC02-02ER63421.

Notes The authors declare that they have no conflicts of interest with the contents of this article.

ACKNOWLEDGMENT

We thank Michael Collazo of the UCLA Crystallization Core for assistance with crystallization screening and the staff of NE-CAT beamline 24-ID-C (NIH-ORIP HEI grant


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S10 RR029205). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC0206CH11357. We thank Joseph Leong for assistance with protein preparation for protein crystallization and Duilio Cascio and David Eisenberg for their support and guidance.

ABBREVIATIONS RuBP, Ribulose 1,5-bisphosphate; RuBisCO – Ribulose 1,5-bisphosphate carboxylase/oxygenase; Ω, CO2/O2 specificity factor; Vc, Vmax for carboxylation; Vo, Vmax for oxygenation; Kc, Km for CO2; Ko, Km for O2; CABP, Carboxyarabinitol 1,5bisphosphate.

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Structural perturbations of Rhodopseudomonas palustris form II

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