Charles A. Fewson

Circadian rhythms in the activity of a plant protein kinase.

Bryophyllum fedtschenkoi is a Crassulacean acid metabolism plant whose phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to a circadian rhythm. A partially purified protein kinase phosphorylated phosphoenolpyruvate carboxylase in vitro with a stoichiometry approaching one per subunit and caused a concomitant 5‐ to 10‐fold decrease in the sensitivity of the carboxylase to inhibition by malate. The sites phosphorylated in vitro were identical to those phosphorylated in intact tissue. The activity of the protein kinase was controlled in a circadian fashion. During normal diurnal cycles, kinase activity appeared between 4 and 5 h after the onset of darkness and disappeared 2‐‐‐‐3 h before the end of darkness. Kinase activity displayed circadian oscillations in constant environmental conditions. The activity of protein phosphatase 2A, which dephosphorylates phosphoenolpyruvate carboxylase, did not oscillate. Treatment of detached leaves with the protein synthesis inhibitors puromycin and cycloheximide blocked the nocturnal appearance of the protein kinase activity, maintained phosphoenolypyruvate carboxylase in the dephosphorylated state and blocked the circadian rhythms of CO2 output that is observed in constant darkness and CO2‐free air. The simplest explanation of the data is that there is a circadian rhythm in the synthesis of phosphoenolpyruvate carboxylase kinase.

Biodegradation of xenobiotic and other persistent compounds: the causes of recalcitrance

Abstract There is enormous scope for increasing the range and extent of biodegradative procedures that can be used to reduce problems of hazardous and unpleasant wastes and to exploit renewable feedstocks. Success will largely depend on a greatly improved understanding of the chemical, biochemical and ecological principles that lead to biological recalcitrance. The other side of the coin is that the same principles are important in understanding and attempting to control biodeterioration.

Changes in the kinetic properties and phosphorylation state of phosphoenolpyruvate carboxylase in Zea mays leaves in reponse to light and dark

In plants such as Zea mays that carry out C4 metabolism, phosphoenolpyruvate carboxylase catalyses the primary fixation of atmospheric CO2. The properties of this enzyme from Z. mays leaves kept in light and in darkness are different. In brightly illuminated leaves, which are actively fixing CO2, the enzyme is less sensitive to feedback inhibition by malate and is phosphorylated on one or more serine residues. In darkened leaves, which are not photosynthesising, the enzyme is more sensitive to inhibition by malate and is much less phosphorylated. This indicates that the activity of the enzyme is controlled by a reversible phosphorylation.

Diurnal changes in the properties of phosphoenolpyruvate carboxylase in Bryophyllum leaves: a possible co valent modification

In plants that show Crassulacean acid metabolism, phosphoenolpyruvate carboxylase catalyses the key step of CO2 fixation at night. We show here that the properties of this enzyme from Bryophyllum fedtschenkoi undergo marked changes between night and day; the night form is much less sensitive to feedback inhibition by malate than is the day form. Incubation of leaves with 32Pi followed by extraction and immunoprecipitation of phosphoenolpyruvate carboxylase showed that only the night form contained 32P. This suggests that the activity of the enzyme is controlled by a covalent modification mechanism.

Bryophyllum fedtschenkoi protein phosphatase type 2A can dephosphorylate phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase, which catalyses the nocturnal fixation of CO2 in Crassulacean acid metabolism (CAM) plants, is regulated by reversible phosphorylation. The phosphorylated ‘night’ form of the enzyme is ten‐fold less sensitive to inhibition by malate than is the dephosphorylated ‘day’ form. The phosphoenolpyruvate carboxylase of the CAM plant Bryophyllum fedtschenkoi can be dephosphorylated by rabbit muscle protein phosphatase type 2A but not by type 1. B. fedtschenkoi leaves contain protein phosphatase activity that can dephosphorylate phosphoenolpyruvate carboxylase. Inhibitor studies show that this enzyme is a type 2A protein phosphatase.

Persistent circadian rhythms in the phosphorylation state of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi leaves and in its sensitivity to inhibition by malate

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPCase) from Bryophyllum fedtschenkoi leaves has previously been shown to exist in two forms in vivo. During the night the enzyme is phosphorylated and relatively insensitive to feedback inhibition by malate whereas during the day the enzyme is dephosphorylated and more sensitive to inhibition by malate. These properties of PEPCase have now been investigated in leaves maintained under constant conditions of temperature and lighting. When leaves were maintained in continuous darkness and CO2-free air at 15°C, PEPCase exhibited a persistent circadian rhythm of interconversion between the two forms. There was a good correlation between periods during which the leaves were fixing respiratory CO2 and periods during which PEPCase was in the form normally observed at night. When leaves were maintained in continuous light and normal air at 15°C, starting at the end of a night or the end of a day, a circadian rhythm of net uptake of CO2 was observed. Only when these constant conditions were applied at the end of a day was a circadian rhythm of interconversions between the two forms of PEPCase observed and the rhythms of enzyme interconversion and CO2 uptake did not correlate in phase or period.

Structure, Distribution and Function of Wax Esters in Acinetobacter calcoaceticus

SUMMARY: The wax esters of Acinetobacter calcoaceticus strains NCIB 8250 and NCIB 10487 harvested at stationary phase from N-limited batch cultures were extracted and shown to consist of C14 to C18 saturated and mono-unsaturated alkan-l-ols randomly esterified with C14 to C18 saturated and mono-unsaturated fatty acids. The mono-unsaturated components contained a cis Δ9 double bond. Wax ester content of strain NCIB 8250 increased under conditions of low growth rate in N-limited continuous culture with carbon and energy source in excess. The high content of wax ester in N-limited cultures of strain NCIB 8250 was lowered by incubation in the absence of a carbon and energy source and the wax ester was converted to water-soluble materials and CO2. It is proposed that in A. calcoaceticus NCIB 8250 the endogenous wax ester present in N-limited cells can serve as an energy reserve. All 19 strains of A. calcoaceticus tested contained some wax ester and as 16 of these strains had increased wax ester contents when harvested from stationary phase N-limited batch cultures, it appears that wax esters are widespread, but not universal, energy storage components in the genus Acinetobacter.

Illumination increases the phosphorylation state of maize leaf phospho enolpyruvate car☐ylase by causing an increase in the activity of a protein kinase

Illumination of maize leaves increases the phosphorylation state of phosphoenolpyruvate carboxylase and reduces the sensitivity of the enzyme to feedback inhibition by malate. Red, white and blue light were each found to be equally potent, and the effect of light was blocked by 3(3,4-dichlorophenyl)-1,1-dimethylurea. A phosphoenolpyruvate carboxylase kinase was partially purified from illuminated maize leaves by a three-step procedure. Phosphorylation of phosphoenolpyruvate carboxylase by this protein kinase reached 0.7-0.8 molecules/subunit and correlated with a 3- to 4-fold increase in Ki for malate. The protein kinase was inhibited by L-malate, but was insensitive to a number of other potential regulators. Freshly prepared and desalted extracts of darkened maize leaves contained very little kinase activity, but the activity appeared when leaves were illuminated for 30-60 min before extraction. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle, but not that of protein phosphatase 1, could dephosphorylate phosphoenolpyruvate carboxylase. The protein phosphatases 1 and 2A activities of maize leaves were not affected by illumination. It is suggested that the major means by which light stimulates the phosphorylation of phosphoenolpyruvate carboxylase is by an increase in the activity of the protein kinase.

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent Ki values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO2 fixation follows. When leaves are maintained in constant darkness at 15 °C until CO2 output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO2 fixation even though the phosphorylation state of PEPCase does not change.

Roles of Circadian Rhythms, Light and Temperature in the Regulation of Phosphoenolpyruvate Carboxylase in Crassulacean Acid Metabolism

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in the leaf tissue of CAM plants. It catalyses the nocturnal fixation of atmospheric CO2 (as HCO3 −) into oxaloacetate, which is subsequently reduced to malate and stored in the vacuole. During the day, malate released from the vacuole is decarboxylated and the resulting CO2 is fixed via the Calvin cycle (e.g. Osmond and Holtum 1981). Consideration of the metabolic pathways involved in CAM suggests that mechanisms must exist to permit flux through PEPC at night and reduce or eliminate it during the day. In this chapter we describe the role of phosphorylation in the regulation of PEPC in the CAM plant Bryophyllum (Kalanchoe) fedtschenkoi.