Akira Wadano

Cytochrome P450 CYP710A Encodes the Sterol C-22 Desaturase in Arabidopsis and Tomato

Δ22-Unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. Here, we describe the identification and characterization of cytochrome P450s belonging to the CYP710A family as the plant C-22 desaturase. Recombinant proteins of CYP710A1 and CYP710A2 from Arabidopsis thaliana and CYP710A11 from tomato (Lycopersicon esculentum) were expressed using a baculovirus/insect system. The Arabidopsis CYP710A1 and tomato CYP710A11 proteins exhibited C-22 desaturase activity with β-sitosterol to produce stigmasterol (CYP710A1, Km = 1.0 μM and kinetic constant [kcat] = 0.53 min−1; CYP710A11, Km = 3.7 μM and kcat = 10 min−1). In Arabidopsis transgenic lines with CYP710A1 and CYP710A11 overexpression, stigmasterol levels increased by 6- to 32-fold. Arabidopsis CYP710A2 was able to produce brassicasterol and stigmasterol from 24-epi-campesterol and β-sitosterol, respectively. Sterol profiling analyses for CYP710A2 overexpression and a T-DNA insertion event into CYP710A2 clearly demonstrated in planta that CYP710A2 was responsible for both brassicasterol and stigmasterol production. Semiquantitative PCR analyses and promoter:β-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the Δ22-unsaturated sterols in Arabidopsis. Our results support the possibility that the CYP710 family may encode P450s of sterol C-22 desaturases in different organisms.

A Bacterial Transgene for Catalase Protects Translation of D1 Protein during Exposure of Salt-Stressed Tobacco Leaves to Strong Light

During photoinhibition of photosystem II (PSII) in cyanobacteria, salt stress inhibits the repair of photodamaged PSII and, in particular, the synthesis of the D1 protein (D1). We investigated the effects of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum ‘Xanthi’) and in transformed plants that harbored the katE gene for catalase from Escherichia coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both wild-type and katE-transformed plants, but the effect of salt stress was less significant in the transformed plants than in wild-type plants. In the presence of lincomycin, which inhibits protein synthesis in chloroplasts, the activity of PSII decreased rapidly and at similar rates in both types of leaf disc during photoinhibition, and the observation suggests that repair of PSII was protected by the transgene-coded enzyme. Incorporation of [35S]methionine into D1 during photoinhibition was inhibited by salt stress, and the transformation mitigated this inhibitory effect. Northern blotting revealed that the level of psbA transcripts was not significantly affected by salt stress or by the transformation. Our results suggest that salt stress enhanced photoinhibition by inhibiting repair of PSII and that the katE transgene increased the resistance of the chloroplasts translational machinery to salt stress by scavenging hydrogen peroxide.

Photosynthetic bioelectrochemical cell utilizing cyanobacteria and water-generating oxidase

A novel photosynthetic bioelectrochemical cell that utilizes biocatalysts in both anode and cathode compartments was constructed for the first time. in the anodic half-cell, some parts of the electrons produced by the oxidation of water in the photosystem of cyanobacteria are transferred to the carbon felt anode through quinonoid electron transfer mediators. The electron is passed to dioxygen to regenerate water in the cathodic half-cell reaction with an aid of bilirubin oxidase reaction via a mediator. The maximum electric power was about 0.3–0.4 W m-2 for the projective electrode surface area at an apparent efficiency of the light energy conversion of 2–2.5%. The factors governing the cell output are discussed on the basis of the potential-current curves of each half-cell.

Purification and partial amino acid sequences of the binding protein from Bombyx mori for CryIAa δ-endotoxin of Bacillus thuringiensis

The binding protein for Bacillus thuringiensis delta-endotoxin, CryIAa, from the brush border membrane of the midgut of Bombyx mori was purified by the dot blot method and delta-endotoxin affinity chromatography. The binding protein was purified to 235-fold enrichment from cholic acid extracts of brush border membranes from B. mori midgut by activated CryIAa-affinity chromatography and DEAE ion-exchange chromatography. The purified binding protein showed a single band of 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and this band specifically reacted to 125I-labeled CryIAa on Immobilon membrane. The affinity of the binding protein for CryIAa was equivalent to that of the brush border membrane vesicles and solubilized membrane proteins. Partial amino acid sequences of the binding protein showed sequence similarity to the cadherin-like binding protein for CryIAb from Manduca sexta, but not for CryIAc binding protein from M. sexta and Heliothis virescens.

Specific Toxicity of δ-Endotoxins from Bacillus thuringiensis to Bombyx mori

Two δ-endotoxins, CryIA(a) and CryIA(b), from Bacillus thuringiensis subsp.. aizawai were used to investigate the specificity in insecticidal activity. CryIA(a) was 17-fold more toxic to Bomhyx mori than CryIA(b). After in vitro solubilization and digestion of these δ-endotoxins, the specificity of toxicity was retained. Trypsin-activated CryIA(a) and CryIA(b) showed specific, high affinity and saturable binding to brush border membrane vesicles (BBMV) from B. mori midguts. These two toxins competed for the same binding site. Dissociation constant for CryIA(a) and CryIA(b) binding to B. mori BBMV was O.89nM and 1.46 nM, respectively. In both toxins, dissociation reaction followed a biphasic process with a fast and a very slow component, suggesting that binding of the toxins proceeds through a reversible component and an apparently irreversible component. In the CryIA(a) dissociation reaction, the irreversible component comprised a large portion of total binding. On the other hand in that of CryIA(b), the reversible component was major. These results suggest that the specific toxicity of the toxins to B. mori may depend mainly on irreversibility.

Electrochemical investigation of cyanobacteria Synechococcus sp. PCC7942-catalyzed photoreduction of exogenous quinones and photoelectrochemical oxidation of water

Abstract The electron transfer from the photosynthetic system in cyanobacteria, Synechococcus sp. PPC7942 to exogenous electron acceptors was examined using several electrochemical techniques. 1,4-Benzoquinone (BQ) and 2,6-dimethyl-1,4-benzoquinone (DMBQ) were found to function as good exogenous electron acceptors for the photosystem. Kinetic analysis with rotating disk amperometry revealed that the photoreduction of these quinones proceeds in Michaelis–Menten type kinetics for the concentration of the quinones and the light intensity. The electron transfer rate of the BQ reduction was as high as 68% compared with that of the photosynthetic oxygen evolution. Synechococcus sp. cell-entrapped and DMBQ-embedded carbon paste electrodes provided steady-state current ascribed to the photoelectrochemical oxidation of water. Although several inhibitors against the photosynthetic system suppressed the photoelectrochemical response, phenylmercury acetate, which inhibits ferredoxin and ferredoxin–NADP oxidoreductase, was found to enhance the photocurrent. Some electrochemical aspects of this system are discussed.

Structure Basis for the Regulation of Glyceraldehyde-3-Phosphate Dehydrogenase Activity via the Intrinsically Disordered Protein CP12

The reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex.

3-hydroxykynurenine as O2⨪ scavenger in the blowfly, Aldrichina grahami

We studied the distribution of O2-.-scavenging activity in 6-day-old larvae of Aldrichina grahami. Total activity was highest in the muscle. The specific activity per milligram of protein in the Malpighian tubules was highest, 10 times the highest elsewhere. Most of the O2-. scavenging activity in muscle depended on superoxide dismutase. However, the activity in the Malpighian tubules mostly depended on 3-hydroxykynurenine.

Studies on nitrogen metabolism in insects: Regulation mechanism of xanthine dehydrogenase in the blowfly, Aldrichina grahami (Diptera; Calliphoridae)

Abstract In adults of A. grahami , xanthine dehydrogenase (EC 1.2.1.37; XDH) activity increased significantly during the first few days after emergence. The activity was high in the fat body, low in the gut, and no activity was detected in the Malipighian tubules. XDH activity increased in relation to the protein content of the diet, and the increase was localized mainly in the fat body. XDH activity increased by 1.5–3 times when the diet contained either amino acids related to purine biosynthesis, or adenine or hypoxanthine. Guanine and xanthine, however, had no effect on XDH activity whilst uric acid lowered the activity. The amino acids and purines which increased XDH activity also augmented the levels of AMP, inosine and hypoxanthine. The increase in XDH activity was due to a de novo synthesis of the enzyme protein.

3-Hydroxykynurenine as a radical scavenger in the blowfly, Aldrichina grahami

Abstract 3-Hydroxykynurenine (3-HOK) accumulated in may tissues of A. grahami, especially in the Malpighian tubules. The compound was present in the insect during its whole life cycle. Its content in the insect seemed to be more than that needed for the synthesis of ommochrome, and was also 5–10-fold of the concentration of other reductants. It can protect the fat body cells of A. grahami from peroxidation and also suppress the decrease of respiration of the cells due to injury by t-butylhydroperoxide (t-BuOOH). The effects of the compound are superior to those of reduced glutathione and uric acid, and approach those of ascorbic acid. The protection of the respiration against injury from t-BuOOH means that 3-HOK effectively scavenged radicals and the more 3-HOK the tissue contained, the less the fat body was injured by t-BuOOH.