A new Escherichia coli laboratory evolution screen for detecting plant ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mutations with enhanced CO2-fixation capacity has identified substitutions that can enhance plant productivity. Selected were a large subunit catalytic (Met-116-Leu) mutation that increases the kcatc of varying plant Rubiscos by 25

Photosynthetic organisms sustain life on Earth by storing solar energy in biomass. Central to this process is rubisco, the enzyme that catalyses the fixation of CO2 to ribulose-1,5-bisphosphate, providing the primary gateway for inorganic carbon into the biosphere. Rubisco′s catalytic efficiency is a major determinant of crop productivity and global carbon flux, making it a longstanding target for protein engineering.1-4 Yet, attempts to enhance its performance through rational design have met limited success due to an incomplete understanding of rubisco′s catalytic mechanism. Here, we report an atomic resolution (1.25 Å) cryo-EM structure of spinach rubisco in complex with the transition-state analogue 2-carboxyarabinitol-1,5-bisphosphate. Supported by large-scale quantum/classical (QM/MM) calculations, our structural analysis reveals protonation equilibria within the active site and unexpected structural flexibility across large protein regions despite the exceptionally tight ligand binding. Our findings provide new insight into the complex interplay of protonation equilibria and conformational sampling, suggesting a novel basis for rubisco′s rational redesign utilizing strategies that rely on a combination of dynamic and electrostatic control. ### Competing Interest Statement S.M. and O.R. are employees of Thermo Fisher Scientific, manufacturer of the Cryo-TEM instrumentation utilised in this study. Swedish Foundation for Strategic Research, https://ror.org/044wr7g58, SwedNESS Kempe Foundation, JCSMK24-599 Swedish Research Council, https://ror.org/03zttf063, 2019-03700_VR, 2023-05296_VR, 2019-01171_Formas, 2020-04081, 2025-04607 Knut and Alice Wallenberg Foundation, 2019.0251, 2024.0220 Göran Gustafsson Foundation Czech Science Foundation, 24-10671S United States Department of Energy, DE-AC02-05CH11231

The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

The activity of the transcription factor EL222 is regulated through protein-chromophore adduct formation, interdomain dynamics, oligomerization and protein-DNA interactions, all triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV/visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using non-canonical amino acids. The observation of only subtle atomic displacements between crystal structures of EL222 with and without blue-light back-illumination, was confirmed by NMR data indicating no major changes in secondary structure and fold compactness. Kinetic experiments in solution provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently-bound N5 protonated, and non-covalently-bound hydroquinone forms of FMN, respectively. Molecular modeling revealed differential dynamics and domain separation times arising from the three FMN states (oxidized, adduct, and reduced). Furthermore, while the dark state is largely monomeric, both lit states undergo slow monomer-dimer exchange. The photoinduced loss of α-helicity, seen by infrared difference spectroscopy, was ascribed to dimeric EL222 species. Unexpectedly, NMR revealed that all three EL222 species (dark, lit1, lit2) can associate with DNA to some extent, but only under illumination a high population of stable complexes is obtained. Overall, we propose a refined model of EL222 photo-activation where photoinduced changes in the oxidation state of FMN and thioadduct formation shift the population equilibrium towards an open conformation that favors self-association and DNA-binding. ### Competing Interest Statement The authors have declared no competing interest.

Improving the carboxylation properties of Rubisco has primarily arisen from unforeseen amino acid substitutions remote from the catalytic site. The unpredictability has frustrated rational design efforts to enhance plant Rubisco towards the prized growth-enhancing carboxylation properties of red algae Griffithsia monilis GmRubisco. To address this, we determined the crystal structure of GmRubisco to 1.7 angstrom. Three structurally divergent domains were identified relative to the red-type bacterial Rhodobacter sphaeroides RsRubisco that, unlike GmRubisco, are expressed in Escherichia coli and plants. Kinetic comparison of 11 RsRubisco chimaeras revealed that incorporating C329A and A332V substitutions from GmRubisco Loop 6 (corresponding to plant residues 328 and 331) into RsRubisco increased the carboxylation rate (k(cat)(c)) by 60%, the carboxylation efficiency in air by 22% and the CO2/O-2 specificity (S-c/o) by 7%. Plastome transformation of this RsRubisco Loop 6 mutant into tobacco enhanced photosynthesis and growth up to twofold over tobacco producing wild-type RsRubisco. Our findings demonstrate the utility of RsRubisco for the identification and in planta testing of amino acid grafts from algal Rubisco that can enhance the enzyme's carboxylase potential.

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length.

Proteins containing the light-oxygen-voltage (LOV) domain are photosensory receptors that mediate biological actions in response to blue light. For instance, the bacterial transcription factor EL222 regulates gene expression in a light-regulated manner and has found use in optogenetic applications. Upon excitation of the embedded flavin mononucleotide (FMN) cofactor, EL222 undergoes structural changes that ultimately drive its association with DNA. However, our knowledge of the light-adapted state(s) and the molecular mechanism underlying the transition between dark and lit states is incomplete. Typically, global protein secondary structure changes and local changes around the FMN chromophore have been detected by Fourier transform infrared (FTIR) spectroscopy (amide I band) and UV-Vis spectroscopy, respectively. To complement these studies, we chose the nitrile stretching vibration as a site-specific infrared reporter of protein microenvironment. EL222 mutants carrying the non-canonical amino acid p-cyanophenylalanine (CNF) were prepared by genetic code expansion technology. We recorded steady-state FTIR difference spectra between irradiated and non-irradiated samples at forty residue positions. Selected EL222 variants were further investigated by time-resolved methods in order to follow the propagation of conformational changes at the FMN, protein backbone (amide I), and protein side-chain (CNF) sites. The varying responses of the CNF tag, which depend on its location, highlights its specificity and sensitivity to evolving amino acid surroundings along the photocycle. Our results emphasize the synergistic combination of protein engineering tools with infrared spectroscopy for delivering site-specific information on light-triggered protein dynamics. Moreover, we show the importance of measuring multiple local and global probes to reveal, with the utmost detail, kinetic pathways in light-sensing proteins.

Structural knowledge of biological macromolecules is essential for understanding their function and for modifying that function by engineering. Protein crystallography is a powerful method for elucidating molecular structures of proteins, but it is essential that the investigator has a basic knowledge of good practices and of the major pitfalls in the technique. Here we describe issues specific for the case of structural studies of strigolactone (SL) receptor structure and function, and in particular the difficulties associated with capturing complexes of SL receptors with the SL hormone ligand in the crystal.

The core of β-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. β-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid α-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form α-aminoadipate semialdehyde) to α-aminoadipic acid, is catalysed by the NAD+-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH from S. clavuligerus in its apo form and in complexes with the cofactor NAD+, the product α-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD+ binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.

Corrections to the article by Daurer et al.[IUCrJ (2017). 4, 251–262] are given.

The possibility of imaging single proteins constitutes an exciting challenge for x-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available x-ray lasers and, as we demonstrate here, contamination of the aerosolized molecules by nonvolatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet size during aerosolization. Here, we show that, with our electrospray injector, we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next-generation x-ray lasers, constitute an important stepping stone toward the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.

The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) fromArabidopsis thalianais reported at 1.5 Å resolution. In light of the importance ofA. thalianaas a model organism for understanding higher plant biology, and the pivotal role of Rubisco in photosynthetic carbon assimilation, there has been a notable absence of anA. thalianaRubisco crystal structure.A. thalianaRubisco is an L8S8hexadecamer comprising eight plastome-encoded catalytic large (L) subunits and eight nuclear-encoded small (S) subunits.A. thalianaproduces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B), and this crystal structure provides a snapshot ofA. thalianaRubisco containing the low-abundance RbcS3B small-subunit isoform. Crystals were obtained in the presence of the transition-state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate.A. thalianaRubisco shares the overall fold characteristic of higher plant Rubiscos, but exhibits an interesting disparity between sequence and structural relatedness to other Rubisco isoforms. These results provide the structural framework to understandA. thalianaRubisco and the potential catalytic differences that could be conferred by alternativeA. thalianaRubisco small-subunit isoforms.

Strigolactones, a group of terpenoid lactones, control many aspects of plant growth and development, but the active forms of these plant hormones and their mode of action at the molecular level are still unknown. The strigolactone protein receptor is unusual because it has been shown to cleave the hormone and supposedly forms a covalent bond with the cleaved hormone fragment. This interaction is suggested to induce a conformational change in the receptor that primes it for subsequent interaction with partners in the signalling pathway. Substantial efforts have been invested into describing the interaction of synthetic strigolactone analogues with the receptor, resulting in a number of crystal structures. This investigation combines a re-evaluation of models in the Protein Data Bank with a search for new conditions that may permit the capture of a receptor-ligand complex. While weak difference density is frequently observed in the binding cavity, possibly due to a low-occupancy compound, the models often contain features not supported by the X-ray data. Thus, at this stage, we do not believe that any detailed deductions about the nature, conformation, or binding mode of the ligand can be made with any confidence.

The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from arctic waters were collected, cultivated, and analyzed for their CO2-fixing capability. We characterized the kinetic properties of five and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL, and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The V-max and K-m for the oxygenase and carboxylase activities at 25 degrees C and the specificity factors (S-c/o) at 15, 25, and 35 degrees C were determined. The S-c/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO(2 )relative to O-2. Structurally, diatom Rubiscos belong to form I C/D, containing small subunits characterized by a short beta A-beta B loop and a C-terminal extension that forms a beta-hairpin structure (beta E-beta F loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxyproline, beta-hydroxyleucine, hydroxylated and nitrosylated cysteine, mono- and dihydroxylated lysine, and trimethylated lysine. Our studies suggest adaptation toward achieving efficient CO2 fixation in arctic diatom Rubiscos.