James N. Burnell

3 - Enzymes of C4 Photosynthesis

[Extract] The history of the resolution of C4 photosynthesis follows a pattern of demonstrating the operation of unique photosynthetic biochemistry by various means and then identifying the enzymes necessary to support that biochemistry. Critical to the developing understanding of this process was the recognition of two types of photosynthetic cells in C4 plants (mesophyll and bundle sheath) with quite different enzyme complements and distinct biochemical roles (see Fig. 3.1). As currently interpreted (see Edwards and Walker, 1983; Hatch, 1987) the reactions unique to the C4 pathway serve, in association with some remarkable modifications of leaf anatomy and ultrastructure, to concentrate CO2 in bundle sheath cells for utilisatiqn by the photosynthetic carbon reduction cycle carboxylase, ribulose L5-bisphosphate carboxylase-oxygenase (Rubisco). The Rubisco-mediated oxygenase reaction and associated photorespiration are thereby eliminated.

Methylglyoxal impairs glucose metabolism and leads to energy depletion in neuronal cells—protection by carbonyl scavengers

Advanced glycation end products (AGEs) are found in various intraneuronal protein deposits such as neurofibrillary tangles in Alzheimers disease and Lewy bodies in Parkinsons disease. Among the many reactive carbonyl compounds and AGE precursors, methylglyoxal is most likely to contribute to intracellular AGE formation, since it is extremely reactive and constantly produced by degradation of triosephosphates. Furthermore, methylglyoxal levels increase under pathophysiological conditions, for example, when trisosephosphate levels are elevated, the expression or activity of glyoxalase I is decreased, as is the case when the concentration of reduced glutathione, the rate-determining co-factor of glyoxalase I, is low. However, the effects of methylglyoxal on mitochondrial function and energy levels have not been studied in detail. In this study, we show that methylglyoxal increases the formation of intracellular reactive oxygen species and lactate in SH-SY5Y neuroblastoma cells. Methylglyoxal also decreases mitochondrial membrane potential and intracellular ATP levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity could contribute to the cytotoxicity of methylglyoxal. The methylglyoxal-induced effects such as ATP depletion and mitochondrial dysfunction can be prevented by pre-incubation of the cells with the carbonyl scavengers aminoguanidine and tenilsetam. In a clinical context, these compounds could not only offer a promising therapeutic strategy to reduce intracellular AGE-accumulation, but also to decrease the dicarbonyl-induced impairment of energy production in aging and neurodegeneration.

Biochemical and molecular characterisation of cubozoan protein toxins.

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as alpha-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: Pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.

Light-dark modulation of leaf pyruvate,Pi dikinase

Abstract Pyruvate,P i dikinase catalyses a unique type of reaction in which two separate substrates are phosphorylated. In leaves this enzyme has a key role in the photosynthetic process known as the C 4 pathway. This article discusses the complex mechanism by which pyruvate,P i dikinase activity is modulated in leaves in response to changing light intensity. Regulation involves interconversion between an active non-phosphorylated form and an inactive phosphorylated form of the enzyme but both reactions proceed by unique mechanisms. The single enzyme catalysing these reactions apparently has two separate active sites.

Regulation of C4 photosynthesis: Purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate,Pi dikinase

Pyruvate,Pi dikinase regulatory protein (PDRP) has been highly purified from maize leaves, and its role in catalyzing both ADP-mediated inactivation (due to phosphorylation of a threonine residue) and Pi-mediated activation (due to dephosphorylation by phosphorolysis) of pyruvate,Pi dikinase has been confirmed. These reactions account for the dark/light-mediated regulation of pyruvate,Pi dikinase observed in the leaves of C4 plants. During purification to apparent homogeneity the ratio of these two activities remained constant. The molecular weight of the native PDRP was about 180,000 at pH 8.3 and 90,000 at pH 7.5. Its monomeric molecular weight was 45,000. It was confirmed that inactive pyruvate,Pi dikinase free of a phosphate group on a catalytic histidine was the preferred substrate for activation. Michaelis constants for orthophosphate and the above form of active pyruvate,Pi dikinase were determined, as well as the mechanism of inhibition of the PDRP-catalyzed reaction by ATP, ADP, AMP, and PPi. For the inactivation reaction, Km values were 1.2 microM for the active pyruvate,Pi dikinase and 52 microM for ADP. CDP and GDP but not UDP could substitute for ADP. The inactivation reaction is inhibited by inactive pyruvate,Pi dikinase competitively with respect to both active pyruvate,Pi dikinase and ADP. Both the activation and inactivation reactions catalyzed by PDRP have a broad pH optimum between 7.8 and 8.3. The results are discussed in terms of the likely mechanism of dark/light regulation of pyruvate,Pi dikinase in vivo.

The Flaveria bidentis β-carbonic anhydrase gene family encodes cytosolic and chloroplastic isoforms demonstrating distinct organ-specific expression patterns

Carbonic anhydrase (CA) catalyzes the interconversion of CO2 and bicarbonate, the forms of inorganic carbon used by the primary carboxylating enzymes of C3 and C4 plants, respectively. Multiple forms of CA are found in both photosynthetic subtypes; however, the number of isoforms and the location and function of each have not been elucidated for any single plant species. Genomic Southern analyses showed that the C4 dicotyledon Flaveria bidentis ‘Kuntze’ contains a small gene family encoding β-CA and cDNAs encoding three distinct β-CAs, named CA1, CA2, and CA3, were isolated. Quantitative reverse transcription-polymerase chain reactions showed that each member of this β-CA family has a specific expression pattern in F. bidentis leaves, roots, and flowers. CA3 transcripts were at least 50 times more abundant than CA2 or CA1 transcripts in leaves. CA2 transcripts were detected in all organs examined and were the most abundant CA transcripts in roots. CA1 mRNA levels were similar to those of CA2 in leaves, but were considerably lower in roots and flowers. In vitro import assays showed CA1 was imported into isolated pea (Pisum sativum) chloroplasts, whereas CA2 and CA3 were not. These results support the following roles for F. bidentis CAs: CA3 is responsible for catalyzing the first step in the C4 pathway in the mesophyll cell cytosol; CA2 provides bicarbonate for anapleurotic reactions involving nonphotosynthetic forms of phosphoenolpyruvate carboxylase in the cytosol of cells in both photosynthetic and nongreen tissues; and CA1 carries out nonphotosynthetic functions demonstrated by C3 chloroplastic β-CAs, including lipid biosynthesis and antioxidant activity.

Dark-light regulation of pyruvate, Pi dikinase in C4 plants: Evidence that the same protein catalyses activation and inactivation

Summary Evidence is provided that the ADP plus ATP-dependent inactivation and the P i -dependent activation of pyruvate, P i dikinase is catalysed by the same protein. Essentially identical behaviour of these two activities was observed during partial purification by (NH 4 ) 2 SO 4 fractionation, affinity chromatography on Sepharose 4B-Blue dextran and filtration on Sepharose 4B. This protein factor, which is sensitive to heat treatment and tryptic digestion, has a molecular weight of approximately 90,000 at pH 7.5 and 180,000 at pH 8.3.

Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri

Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of approximately 370 and 145kDa, and one minor cytolysin ( approximately 70kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits ( approximately 43 and 45kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145kDa haemolysin predominately contains two other major proteins ( approximately 39 and 41kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase ( approximately 6-7min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom.

Cloning and characterization of Escherichia coli DUF299: a bifunctional ADP-dependent kinase - Pi-dependent pyrophosphorylase from bacteria

BackgroundPhosphoenolpyruvate synthetase (PEPS; EC 2.7.9.2) catalyzes the synthesis of phosphoenolpyruvate from pyruvate in Escherichia coli when cells are grown on a three carbon source. It also catalyses the anabolic conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis. A bioinformatics search conducted following the successful cloning and expression of maize leaf pyruvate, orthophosphate dikinase regulatory protein (PDRP) revealed the presence of PDRP homologs in more than 300 bacterial species; the PDRP homolog was identified as DUF299.ResultsThis paper describes the cloning and expression of both PEPS and DUF299 from E. coli and establishes that E. coli DUF299 catalyzes both the ADP-dependent inactivation and the Pi-dependent activation of PEPS.ConclusionThis paper represents the first report of a bifunctional regulatory enzyme catalysing an ADP-dependent phosphorylation and a Pi-dependent pyrophosphorylation reaction in bacteria.