Enzymatic O
sensors transduce the availability of O
within the cell into a physiological, typically adaptive response. One such O
-sensing enzymatic family is the N-terminal cysteine dioxygenases in ...plants (plant cysteine oxidases PCOs). In vitro kinetic studies have determined the O
-sensing capacity of PCOs. Here we describe the rationale and experimental protocol for an assay with which the O
sensitivity of Arabidopsis thaliana PCOs (AtPCOs) can be measured. We explain each step from the recombinant protein synthesis of AtPCOs to the steady-state kinetic assays of AtPCOs for primary substrate and O
from which kinetic parameters can be derived. The same techniques can be applied to other N-terminal cysteine thiol dioxygenases, e.g. 2-aminoethanethiol dioxygenase (ADO), and similar principles can be applied to determine kinetic characteristics of other oxygenase enzymes towards O
.
Regulation of the hypoxic response in humans is regulated by the post-translational hydroxylation of hypoxia inducible transcription factor; a recombinant form of a human prolyl-4-hydroxylase (PHD2) ...was characterised and shown to have an unexpectedly high affinity for, and to copurify with endogenous levels of, its Fe(ii) cofactor and 2-oxoglutarate cosubstrate.
Cysteine dioxygenases, 3‐mercaptopropionate dioxygenases and mercaptosuccinate dioxygenases are all thiol dioxygenases (TDOs) that catalyse oxidation of thiol molecules to sulphinates. They are ...Fe(II)‐dependent dioxygenases with a cupin fold that supports a 3xHis metal‐coordinating triad at the active site. They also have other, broadly common features including arginine residues involved in substrate carboxylate binding and a conserved trio of residues at the active site featuring a tyrosine important in substrate binding catalysis. Recently, N‐terminal cysteinyl dioxygenase enzymes (NCOs) have been identified in plants (plant cysteine oxidases, PCOs), while human 2‐aminoethanethiol dioxygenase (ADO) has been shown to act as both an NCO and a small molecule TDO. Although the cupin fold and 3xHis Fe(II)‐binding triad seen in the small molecule TDOs are conserved in NCOs, other active site features and aspects of the overall protein architecture are quite different. Furthermore, the PCOs and ADO appear to act as biological O2 sensors, as shown by kinetic analyses and hypoxic regulation of the stability of their biological targets (N‐terminal cysteine oxidation triggers protein degradation via the N‐degron pathway). Here, we discuss the emergence of these two subclasses of TDO including structural features that could dictate their ability to bind small molecule or polypeptide substrates. These structural features may also underpin the O2‐sensing capability of the NCOs. Understanding how these enzymes interact with their substrates, including O2, could reveal strategies to manipulate their activity, relevant to hypoxic disease states and plant adaptive responses to flooding.
Thiol dioxygenases (TDOs) catalyse oxidation of thiols to sulphinates. Recent reports identify TDOs which catalyse oxidation of N‐terminal cysteine residues of protein substrates in an O2‐sensitive manner, promoting degradation: N‐terminal cysteinyl dioxygenases (NCOs). These appear to form a separate subclass of TDO from those which catalyse oxidation of small molecule thiols. We discuss the structural differences between these two TDO subclasses and suggest how they link to function.
SUMMARY
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.
Significance Statement
Plant cysteine oxidase enzymes sense oxygen availability such that when oxygen levels drop, as occurs when plants are flooded, they initiate an adaptive response to help the plant survive until oxygen becomes available again. Manipulating these enzymes to alter their oxygen‐sensing and/or substrate‐binding characteristics has the potential to prolong flood tolerance in plants, illustrating that enzyme engineering could be adopted more widely as a strategy to improve plant stress resistance and productivity.
N/O-methylation of DNA can be cytotoxic and mutagenic; therefore, enzymes that reverse DNA methylation are essential for organism survival. Several 2-oxoglutarate-dependent oxygenases and ...methyltransferases that remove a methyl group from a methylated DNA base have been identified. Studies of their kinetics and search for their inhibitors have been retarded by the lack of an approach to directly quantitate DNA substrates and products that differ by a single methyl group. Here, we introduce such an approach, which is based on capillary electrophoresis with laser-induced fluorescence detection. We achieved baseline separation of a fluorescently labeled 15-nucleotide-long single-base methylated DNA substrate from its demethylated product, followed by its quantitative detection. We then used this approach to study the kinetics of AlkB-catalyzed DNA demethylation and screen a number of potential inhibitors of this reaction. Ten new inhibitors, which can be used as templates in developing therapies targeting AlkB-like enzymes, were identified. Our approach will be applicable for in vitro kinetic studies of known DNA demethylating and methylating enzymes and in the discovery of new ones.
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.
Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading ...to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.
In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to ...activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.
Myosin binding protein-C (MyBP-C) is a thick filament–associated protein localized to the crossbridge-containing C zones of striated muscle sarcomeres. The cardiac isoform is composed of eight ...immunoglobulin I–like domains and three fibronectin 3–like domains and is known to be a physiological substrate of cAMP-dependent protein kinase. MyBP-C contributes to thick filament structure via interactions at its C-terminus with the light meromyosin section of the myosin rod and with titin. The protein also has a role in the regulation of contraction, due to the binding of its N-terminus to the subfragment-2 portion of myosin, which reduces actomyosin ATPase activity; phosphorylation abolishes this interaction, resulting in release of the “brake” on crossbridge cycling. Several structural models of the interaction of MyBP-C with myosin have been proposed, although its precise arrangement on the thick filament remains to be elucidated. Mutations in the gene encoding cardiac MyBP-C are a common cause of hypertrophic cardiomyopathy, and this has led to increased interest in the proteinʼs function. Investigation of disease-causing mutations in domains with unknown function has led to further insights into the mechanism of cMyBP-C action. This Review aims to collate the published data on those aspects of MyBP-C that are well characterized and to consider new and emerging data that further define its structural and regulatory roles and its arrangement in the sarcomere. We also speculate on the mechanisms by which hypertrophic cardiomyopathy–causing truncation and missense mutations affect the normal functioning of the sarcomere.
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