Kazumori Masamoto

Detailed biosynthetic pathway to decaprenoxanthin diglucoside in Corynebacterium glutamicum and identification of novel intermediates

Abstract. Carotenogenic mutants of Corynebacterium glutamicum were analyzed for their carotenoid content. Mutant MV10 accumulated the same carotenoids as the wild-type, decaprenoxanthin, decaprenoxanthin monoglucoside, and (2R,6R,2′R,6′R)-decaprenoxanthin di-(β-D)-glucoside, but in three-fold higher amounts. In addition, decaprenoxanthin diglucoside fatty acid esters and the intermediates nonaprene, 2-(3-methyl-2-butenyl)-ε,ψ-carotene, and sarcinene, 2,2′-bis(3-methyl-2-butenyl)-ε,ε-carotene were identified as minor carotenoids. The pink mutants MV40 and MV60 synthesized only lycopene. From another pink mutant, MV70, novel C50-carotenoids were isolated. By NMR and mass spectroscopy, nonaflavuxanthin, 2-(4-hydroxy-3-methyl-2-butenyl)-1,16-didehydro-1,2-dihydro-ψ,ψ-carotene, and flavuxanthin, 2,2′-bis(4-hydroxy-3-methyl-2-butenyl)-1,16,1′,16′-tetradehydro-1,2,1′,2′-tetrahydro-ψ,ψ-carotene, were identified. The identification of these intermediates revealed the detailed pathway for the formation of decaprenoxanthin derivatives in Corynebacterium glutamicum.

Aluminum Tolerance Acquired during Phosphate Starvation in Cultured Tobacco Cells

Al toxicity in cultured tobacco cells (Nicotiana tabacum L. cv Samsun; nonchlorophyllic cell line SL) has been investigated in nutrient medium. In this system, Al and Fe(II) (ferrous ion) in the medium synergistically result in the accumulation of both Al and Fe, the peroxidation of lipids, and eventually death in cells at the logarithmic phase of growth (+P cells). A lipophilic antioxidant, N,N[prime]-diphenyl-p-phenylenediamine, protected +P cells from the peroxidation of lipids and from cell death, suggesting that a relationship exists between the two. Compared with +P cells, cells that had been starved of Pi (-P cells) were more tolerant to Al, accumulated 30 to 40% less Al and 70 to 90% less Fe, and did not show any evidence of the peroxidation of lipids during Al treatment. These results suggest that -P cells exhibit Al tolerance because their plasma membranes are protected from the peroxidation of lipids caused by the combination of Al and Fe(II). It seems likely that the exclusion of Fe from -P cells might suppress directly Fe-mediated peroxidation of lipids. Furthermore, since -P cells accumulated [beta]-carotene, it is proposed that this carotenoid pigment might function as a radical-trapping antioxidant in the plasma membrane of cells starved of Pi.

The leaves of the common box, Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid, anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,1H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.

Accumulation of Zeaxanthin in cells of the cyanobacterium, Synechococcus sp. Strain PCC 7942 grown under high irradiance

Summary An analytical study was made of the carotenoid composition of the cyanobacterium, Synechococcus sp. strain PCC 7942, using reversed-phase HPLC. Zeaxanthin increased in cells grown under high irradiance (high light, HL) by 3.9-fold (cell amount basis) and 4.2-fold (chlorophyll a basis), when compared with that of cells grown under low irradiance. The low-temperature-induced absorbance increase at 390 nm of the HL-cells suggested an accumulation of zeaxanthin in the cytoplasmic membranes.

Accumulation of zeaxanthin in cytoplasmic membranes of the Cyanobacterium Synechococcus sp. strain PCC7942 grown under high light condition

Summary The exposure of cells to excess light enhanced the content of zeaxanthin in the cells of the Cyanobacte- rium, Synechococcus sp. strain PCC7942. The isolated cytoplasmic membranes of the high-light grown cells contained 2.7 times more zeaxanthin, on a protein basis, than low-light grown cells. However, the zeaxanthin content in the thylakoid membranes was not affected significantly. The accumulation of zea- xanthin in the cytoplasmic membranes under high-light conditions indicates a mechanism that can protect the cells against high-light exposure.

Electrical potential changes in the surface and the central region of chromatophore membranes of photosynthetic bacteria detected by the absorbance changes of ethidium and carotenoid

Abstract (1) Changes of local intramembrane electrical field in the surface and central region of the chromatophore membrane during energization were studied both by the measurement of absorbance changes of ethidium, a monovalent cationic dye, and of carotenoid, the intrinsic probe of electrical field. (2) Binding of ethidium to the chromatophore membrane of Rhodopseudomonas sphaeroides was found to be dependent on the energization of membrane as well as on the ionic condition of the medium. The dye was released from the membrane when salt was added to the suspension, indicating the electrostatic interaction between the positive dye and the net negative membrane surface. The result was explained by the surface-potential dependent distribution of the dye to the membrane surface, as seen with other charged dyes (Masamoto, K., Matsuura, K., Itoh, S. and Nishimura, M. (1981) Biophys. Acta 638, 108–115). (3) Energization of chromatophores by flash-light-induced absorbance change of ethidium showing a similar difference spectrum to that induced by the addition of salts. The release of ethidium by a single turn-over flash of saturating intensity was estimated to be 0.22 ethidium per reaction center. Addition of ethidium (at 200 μM) slightly affected the flash-induced absorbance change of carotenoid which responds to the intramembrane electricalfield change, indicating a low-membrane permeability of the dye. The extent of the absorbance change of ethidium was linear to that of carotenoid, and was abolished in the presence of valinomycin plus K + . However, the rise and decay kinetics of the absorbance change of ethidium was different from that of carotenoid. (4) These absorbance changes of ethidium and carotenoid can be explained by a model in which ethidium responds to the potential changes in the surface region and carotenoid in the central hydrophobic region of the chromatophore membrane.

Phosphatidylglycerol an essential structural and functional constituent of photosynthetic membrane

The role of phosphatidylglycerol (PG) was studied in a Synechocystis sp. PCC6803 mutant to synthesize PG. The PG content in the membranes was controlled by the externally added PG. With the loss of PG content we observed a decrease in the photosynthetic oxygen evolving activity. It was accompanied with a significant decrease in the chlorophyll content of the cells. The change in photosynthetic activity was caused by the inhibition of electron transfer on the donor side between QA and QB plastoquinones. It was measured by oxygen evolution, induction of chlorophyll fluorescence, change in fast fluorescence yield detected by double flash technique and thermoluminescence. In the absence of PG, oxygen evolution was inhibited by the addition of artificial quinones. The altered fluorescence induction suggested the inactivation of the QB function in the PG-depleted cells. A fast change in fluorescence yield was indicated by slow (20 ms) reduction of P680+, and a very slow re-oxidation (1s) of QA. Thermoluminescence measurement also confirmed the inhibition of electron transfer from QA- to QB in the PG-depleted cells and suggested the accumulation of Yz+ and His+ in addition to S2 state. No significant change of redox level of QA and S2 state was detected. These findings demonstrate that loss of PG leads to the (1) inactivation of the QB function, (2) inhibition of the turnover of water splitting reaction and (3) modification of energy transfer from antenna to reaction center complex. Thus PG molecules seem to be indispensable structural components of the active PS II reaction center.

Characterization of a Temperature-Conditional Arabidopsis Mutant Defective in D-1-Deoxyxylulose-5-Phosphate Synthase

Isoprenoids comprise the largest family of natural products, including a variety of essential components in plants, such as hormones (gibberellin, abscisic acid), photosynthetic pigments (chlorophylls, carotenoids), an electron carrier (plastquinone) and the structural components of membranes (phytosterols) (1). It was generally assumed that isopentenyl diphosphate (IPP), which is a common precursor of all isoprenoids, was synthesized through the acetate/mevalonate pathway. Recent investigations, however, have demonstrated that in bacteria, higher plants and algae there exists a mevalonate-independent pathway for IPP biosynthesis (2,3). At present, its biological functions and regulatory mechanisms in higher plants are still largely unknown. The first reaction of this mevalonate-independent pathway consists of the condensation of hydroxyethyl thiamine derived from pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate (DXP) (4). In Escherichia coli, the gene encoding DXP synthase (dxs) has been identified and characterized in detail (5). In this report, we characterized a temperature-conditional chlorotic mutant of Arabidopsis thaliana, chs5 (6). Mapping and sequence analysis showed that chs5 is allelic to the previously isolated albino mutant, clal, which encodes an Arabidopsis homologue of dxs. We identified the single-base pair substition of the CLA1 gene in the chs5 mutant, which changes aspartate (627) to asparagine residue. This residue is invariant in the dxs gene family, suggesting that the residue has an important role to play in this gene family’s activity.