Two amino acids help regulate an enzyme that clears Rubisco inhibitors

Rubisco helps plants turn carbon dioxide into the organic compounds needed for growth. But its activity depends on more than how much of the enzyme a leaf contains. Certain sugar phosphates can bind tightly to Rubisco and prevent it from working. In many plant species, these inhibitors accumulate during darkness or prolonged low light.

One enzyme involved in clearing them is CA1Pase, short for 2-carboxy-D-arabinitol 1-phosphate phosphatase. It removes phosphate groups from certain inhibitors after another protein, Rubisco activase, releases them from Rubisco. Understanding how CA1Pase itself is controlled could help explain differences in Rubisco regulation among plants.

Connor Nehls-Ramos, Elizabete Carmo-Silva and Douglas J. Orr at Lancaster University investigated this enzyme in cowpea. Their study, posted on bioRxiv on November 9, 2026, is a preprint that has not been peer reviewed. It combines protein-sequence comparisons, computer-predicted structures and laboratory experiments with proteins produced in bacteria—not tests of modified crops.

A regulatory role for the enzyme’s second half

CA1Pase has two main sections, or domains. Its first domain contains the amino acids needed to carry out phosphate removal. The role of the second, called the C domain, has been less clear.

The researchers found that the predicted shapes of the two domains resemble each other, although the cowpea C domain lacks most of the amino acids normally required for this catalytic reaction. They interpret it as a regulatory domain rather than a second working catalytic site.

Within this region, they identified two cysteine amino acids, numbered 292 and 297, separated by four other amino acids. Cysteines can participate in reversible chemical changes that make proteins sensitive to their surroundings. The pair was predicted to form a disulphide bond, a chemical connection between their sulfur-containing side chains.

This suggested a possible connection to redox regulation: control through oxidation and reduction, reactions involving electron transfer. Inside chloroplasts, conditions generally become more reducing during active photosynthesis and more oxidising in darkness.

Changing the cysteines changed the response

To test the pair’s role, the team produced the normal cowpea enzyme alongside versions in which either cysteine, or both, had been replaced with alanine. They measured phosphate removal across mixtures of reduced and oxidised glutathione, a molecule involved in cellular redox chemistry.

The assays used CRBP, an alternative substrate previously used in CA1Pase studies, rather than the nighttime inhibitor CA1P itself. This distinction matters: the experiments measured enzyme activity under laboratory conditions, not inhibitor clearance inside a leaf.

The normal enzyme became more active as conditions became more reducing. All three altered versions had lower activity than the normal enzyme under fully reducing conditions. Replacing cysteine 297, alone or together with cysteine 292, reversed the usual response at the fully reduced end of the experiment. The version lacking only cysteine 292 still increased in activity as conditions became more reducing.

The results therefore implicate the pair in redox control, but do not reveal a simple switch whose removal always leaves the enzyme active.

Protein pairing offers a possible explanation

Computer models suggested that two CA1Pase molecules could associate as a dimer, with the cysteine pair near their predicted contact surface. Experiments separating proteins by their behaviour in a chromatography column also suggested the presence of single molecules and dimers. Reducing conditions appeared to favour dimers, and measurements of separated fractions supported greater activity in the proposed dimer form.

These assignments remain provisional. The apparent protein sizes differed from those expected, and chromatography estimates can depend on protein shape. No experimentally resolved CA1Pase structure was available to establish the proposed arrangement or its redox-dependent changes.

A species comparison, not proof of causation

The researchers also examined 15 legume species with both CA1Pase sequences and previously reported dark-inhibition data. Five had serine or threonine replacing one or both key cysteines. All five showed low accumulation of dark inhibitors.

One possibility is that these natural variants continue degrading inhibitors under dark, oxidising conditions. But the comparison does not establish that mechanism, and the natural substitutions differ from the alanine replacements tested in the laboratory. Information about enzyme expression and abundance in these species is also limited.

The findings identify a specific regulatory feature worth investigating. They do not demonstrate improved photosynthesis, stress tolerance or crop yield. Establishing those consequences will require experiments in plants, alongside direct structural work to test how the cysteines influence enzyme pairing and activity.