Gernot Schultz

The formation of homogentisate in the biosynthesis of tocopherol and plastoquinone in spinach chloroplasts

AbstractHomogentisate is the precursor in the biosynthesis of α-tocopherol and plastoquinone-9 in chloroplasts. It is formed of 4-hydroxyphenylpyruvate of the shikimate pathway by the 4-hydroxyphenylpyruvate dioxygenase. In experiments with spinach the dioxygenase was shown to be localized predominatedly in the chloroplasts. Envelope membranes exhibit the highest specific activity, however, because of the high stromal portion of chloroplasts, 60–80% of the total activity is housed in the stroma. The incorporation of 4-hydroxyphenylpyruvate into 2-methyl-6-phytylquinol as the first intermediate in the tocopherol synthesis by the two-step-reaction: 4-Hydroxyphenylpyruvate → Homogentisaten

The plastidic 3-phosphoglycerate → acetyl-CoA pathway in barley leaves and its involvement in the synthesis of amino acids, plastidic isoprenoids and fatty acids during chloroplast development

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Uptake of shikimate pathway intermediates by intact chloroplasts

n 2-Methyl-6-phytylquinol was demonstrated by using envelope membranes. Homogentisate originates directly from 4-hydroxyphenylpyruvate of the shikimate pathway. Additionally, a bypass exists in chloroplasts which forms 4-hydroxyphenylpyruvate from tyrosine by an L-amino-acid oxidase of the thylakoids and in peroxisomes by a transaminase reaction. Former results about the dioxygenase in peroxisomes were verified.

Synthesis of Plastoquinone-9, α-Tocopherol and Phylloquinone (Vitamin K1) and Its Integration in Chloroplast Carbon Metabolism of Higher Plants

Abstract Geranylgeranyl substituted methylquinols are shown to be precursors of tocopherol biosynthesis in spinach chloroplasts as well as phytyl substituted ones. The geranylgeranyl substituted quinols are methylated even to a greater extent than the phytyl substituted ones. The connection to the so far known biosynthetic origin of α-tocopherol is probably -tocotrienol which is hydrogenated to γ-tocopherol and then further methylated to α-tocopherol.

Control of Shikimate Pathway in Spinach Chloroplasts by Exogenous Substrates

Earlier studies on the synthesis of C3-derived amino acids, plastidic isoprenoids and fatty acids from CO2 by isolated chloroplasts in the light indicate the presence of a complete, but low-capacity, chloroplast (chlp) 3-phosphoglycerate → acetyl-CoA pathway which is predominantely active in immature (developing) chloroplasts (A. Heintze et al., 1990, Plant Physiol. 93, 1121–1127). In this paper, we demonstrate the activity of the enzymes involved i.e. chlp phosphoglycerate mutase, chlp enolase, chlp pyruvate kinase and chlp pyruvate-dehydrogenase complex (PDC), in the stroma of purified barley (Hordeum sativum L.) chloroplasts of different developmental stages. The chlp phosphoglycerate mutase was partially purified for the first time. The activities of the enzymes of this chlp pathway (except PDC) were about a magnitude lower than those of the cytosolic enzymes. The chlp PDC of barley was more active than that of spinach. The apparent Km values of the enzymes of this pathway were about 100 μM or lower except for the chlp phosphoglycerate mutase which had a Km of 1.6–1.8 mM for 3-phospho-d-glycerate. Interestingly, no appreciable change in the activity of these enzymes was observed during maturation of the chloroplasts. In contrast, the activity of the reversible NADP+-glyceraldehyde 3-phosphate dehydrogenase increased about five times (from 140 to 590 nkat per g leaf dry weight). The following hypothesis is put forward to explain the regulation of carbon metabolism during chloroplast development: 3-phospho-d-glycerate is withdrawn from a common pool by the actions of 3-phosphoglycerate kinase and NADP+-glyceraldehyde-3-phosphate dehydrogenase, the activity of which increases considerably during maturation of chloroplasts. This leads to an insufficient supply of 3-phospho-glycerate for the chlp phosphoglycerate mutase, which has a low affinity for its substrate.

Competition of CO2 and Acetate as Substrates for Fatty Acid Synthesis in Immature Chloroplasts of Barley Seedlings

The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the Km values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. Vmax for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)-1·h-1. The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 μM) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (ΔpH) and the transmembrane potential (ΔΨ).

Synthesis of prenylquinones in chloroplasts

Abstract In intact chloroplasts isolated from barley, the rate of aromatic amino acid synthesis from 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS) or shikimate (SKA), 2 mM each, and [1- 14 C]phosphoenolpyruvate (PEP) was 2.5-, 6- and 2.5-fold compared with the rate from photosynthetically fixed CO 2 and [1- 14 C]PEP. The same enhancement was found for protoplasts from barley. Using DHS (2 mM), maximal rates for aromatic amino acid synthesis of about 800 nmol carbon incorporated mg −1 chlorophyll hr −1 were obtained for chloroplasts. Further support for a specific import of compounds mentioned above was given by isotopic dilution experiments using [G- 14 C]SKA and unlabelled DHQ, DHS and quinate, respectively. The transfer of these pre-aromatic acids across the chloroplast envelope membrane is discussed from the point of view of an intra- and intercellular transport of shikimate pathway intermediates during plant development.

Non-light-dependent shikimate pathway in plastids from pea roots

Plastoquinone-9, α-tocopherol and phylloquinone are known as plastidic prenylquinones fulfilling important functions: Plastoquinone-9 acts as mobile electron and proton carrier in photosynthetic electron transport and is involved in building up the electrochemical proton potential at the chloroplast cytochrom b6/f complex /1, 2/. α-Tocopherol is involved in inactivating energized oxygen species, formed in the light, by scavenging radicals and quenching singlett oxygen /3/. Phylloquinone is known as obligatory constituent of PS I (K1 /chlorophyll of PS I ratio about 1: 100 /4/).