A map of the rubisco biochemical landscape.

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

Deep-branching evolutionary intermediates reveal structural origins of form I rubisco.

The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme-found in plants, algae, and cyanobacteria-forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I') has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I' rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I'' rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I'' rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution.

Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae.

All aerobic organisms require O2 for survival. When their O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O2-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O2 availability and can therefore act as plant O2 sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, Marchantia polymorpha (MpPCO), and the freshwater algae, Klebsormidium nitens (KnPCO), have a similar function as PCO enzymes from Arabidopsis thaliana. We report that MpPCO and KnPCO show O2-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the Arabidopsis ERF-VIIs transcription factors. This work confirms functional and O2-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O2-sensing pathways in these organisms and suggesting PCO O2-sensing function could be important throughout the plant kingdom.

Targeting plant cysteine oxidase activity for improved submergence tolerance.

Plant cysteine oxidases (PCOs) are plant O2 -sensing enzymes. They catalyse the O2 -dependent step which initiates the proteasomal degradation of Group VII ethylene response transcription factors (ERF-VIIs) via the N-degron pathway. When submerged, plants experience a reduction in O2 availability; PCO activity therefore decreases and the consequent ERF-VII stabilisation leads to upregulation of hypoxia-responsive genes which enable adaptation to low O2 conditions. Resulting adaptations include entering an anaerobic quiescent state to maintain energy reserves and rapid growth to escape floodwater and allow O2 transport to submerged tissues. Stabilisation of ERF-VIIs has been linked to improved survival post-submergence in Arabidopsis, rice (Oryza sativa) and barley (Hordeum vulgare). Due to climate change and increasing flooding events, there is an interest in manipulating the PCO/ERF-VII interaction as a method of improving yields in flood-intolerant crops. An effective way of achieving this may be through PCO inhibition; however, complete ablation of PCO activity is detrimental to growth and phenotype, likely due to other PCO-mediated roles. Targeting PCOs will therefore require either temporary chemical inhibition or careful engineering of the enzyme structure to manipulate their O2 sensitivity and/or substrate specificity. Sufficient PCO structural and functional information should make this possible, given the potential to engineer site-directed mutagenesis in vivo using CRISPR-mediated base editing. Here, we discuss the knowledge still required for rational manipulation of PCOs to achieve ERF-VII stabilisation without a yield penalty. We also take inspiration from the biocatalysis field to consider how enzyme engineering could be accelerated as a wider strategy to improve plant stress tolerance and productivity.

The plant cysteine oxidases from Arabidopsis thaliana are kinetically tailored to act as oxygen sensors.

Group VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants. ERF-VII stability is controlled in an O2-dependent manner by the Cys/Arg branch of the N-end rule pathway whereby oxidation of a conserved N-terminal cysteine residue initiates target degradation. This oxidation is catalyzed by plant cysteine oxidases (PCOs), which use O2 as cosubstrate to generate Cys-sulfinic acid. The PCOs directly link O2 availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O2 sensors. However, their ability to respond to fluctuations in O2 concentration has not been established. Here, we investigated the steady-state kinetics of Arabidopsis thaliana PCOs 1-5 to ascertain whether their activities are sensitive to O2 levels. We found that the most catalytically competent isoform is AtPCO4, both in terms of responding to O2 and oxidizing AtRAP2.2/2,12 (two of the most prominent ERF-VIIs responsible for promoting the hypoxic response), which suggests that AtPCO4 plays a central role in ERF-VII regulation. Furthermore, we found that AtPCO activity is susceptible to decreases in pH and that the hypoxia-inducible AtPCOs 1/2 and the noninducible AtPCOs 4/5 have discrete AtERF-VII substrate preferences. Pertinently, the AtPCOs had Km(O2)app values in a physiologically relevant range, which should enable them to sensitively react to changes in O2 availability. This work validates an O2-sensing role for the PCOs and suggests that differences in expression pattern, ERF-VII selectivity, and catalytic capability may enable the different isoforms to have distinct biological functions. Individual PCOs could therefore be targeted to manipulate ERF-VII levels and improve stress tolerance in plants.

Preparation and characterisation of high-density ionic liquids incorporating halobismuthate anions.

A range of ionic liquids containing dialkylimidazolium cations and halobismuthate anions ([BiBr(x)Cl(y)I(z)](-) and [Bi2Br(x)Cl(y)I(z)](-)) were synthesised by combining dialkylimidazolium halide ionic liquids with bismuth(III) halide salts. The majority were room temperature liquids, all with very high densities. The neat ionic liquids and their mixtures with 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide were characterised using Densitometry, Viscometry, NMR Spectroscopy, Electrospray Ionisation Mass Spectrometry (ESI), Liquid Secondary Ion Mass Spectrometry (LSIMS), Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (MALDI), X-Ray Photoelectron Spectroscopy (XPS) and Thermogravimetric Analysis (TGA), to establish their speciation and suitability for high-temperature applications.