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H Kleinig

Publications and source records attributed to H Kleinig.

At least 19 recordsLinked to original sources
PubMed2000

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis.

During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakolds which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents.

Alkyl and Aryl Transferases↗
PubMed1998

Induced beta-carotene synthesis driven by triacylglycerol deposition in the unicellular alga dunaliella bardawil

Under stress conditions such as high light intensity or nutrient starvation, cells of the unicellular alga Dunaliella bardawil overproduce beta-carotene, which is accumulated in the plastids in newly formed triacylglycerol droplets. We report here that the formation of these sequestering structures and beta-carotene are interdependent. When the synthesis of triacylglycerol is blocked, the overproduction of beta-carotene is also inhibited. During overproduction of beta-carotene no up-regulation of phytoene synthase or phytoene desaturase is observed on the transcriptional or translational level, whereas at the same time acetyl-CoA carboxylase, the key regulatory enzyme of acyl lipid biosynthesis, is increased, at least in its enzymatic activity. We conclude that under normal conditions the carotenogenic pathway is not maximally active and may be appreciably stimulated in the presence of sequestering structures, creating a plastid-localized sink for the end product of the carotenoid biosynthetic pathway.

Journal Article↗
PubMed1997

Chloroplast import of four carotenoid biosynthetic enzymes in vitro reveals differential fates prior to membrane binding and oligomeric assembly.

The precursor proteins of the carotenogenic enzymes geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclase were imported into isolated pea chloroplasts. Geranylgeranyl diphosphate synthase remained soluble in the stroma in a free form and phytoene synthase associated to thylakoid membranes upon import, both as expected. Surprisingly, phytoene desaturase and lycopene cyclase, which strongly depend on membrane association for enzymatic activity, also remained soluble in the chloroplast stroma. The soluble forms of these enzymes were, however, still competent for membrane-association, e.g. with protein-free liposomal membranes. Indeed the soluble forms of phytoene synthase, phytoene desaturase and lycopene cyclase occurred as ATP- and cold-sensitive high-molecular-mass complexes. Gel-filtration experiments and blue native-PAGE plus autoradiography and western blot analysis indicated a participation of the chloroplast 60-kDa chaperonin (Cpn60) in the soluble high-molecular-mass complexes of imported carotenogenic enzymes. Finally, it was inferred that a membrane-bound regulatory factor plays a decisive role in membrane-binding.

Adenosine Triphosphate↗
PubMed1997

Light-dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings.

In chloroplasts, carotenoids are essential pigments involved in photosynthesis. During-photomorphogenesis, a coordinated increase in the amounts of chlorophylls and carotenoids, in conjugation with other components, leads to the formation of a functional photosynthetic apparatus. To investigate the regulation of carotenoid biosynthesis during this process at the molecular level, GGPS, PSY and PDS cDNAs have been cloned from white mustard (Sinapis alba L). GGPS encodes a key enzyme in plastid isoprenoid metabolism, while the products of PSY and PDS catalyse the subsequent steps in carotenoid biosynthesis. Due to the low mRNA levels of the genes involved, the use of a RT-PCR protocol was necessary to measure gene expression during photomorphogenesis. With light, there is an up-regulation of PSY expression, the first gene within the carotenoid biosynthetic pathway, while PDS and GGPS expression levels remain constant. Treatment with different light qualities reveals a phytochrome-mediated regulation of PSY expression in developing white mustard seedlings. To obtain more detailed information on the light-regulation, Arabidopsis thaliana wild-type and phytochrome mutants were utilized. Continuous far-red and red light both increase the expression of PSY in wild-type seedlings, demonstrating that both light-labile and light-stable phytochromes are involved in PSY regulation. The response to far-red light is completely abolished in the phyA mutant, showing that PHYA mediates the increase in PSY transcript levels under these light conditions. In the phyB mutant, the red light response is normal, indicating that PSY expression is not controlled by PHYB but by other light-stable phytochromes. Measurement of chlorophylls and carotenoids under the same light regimes shows that the up-regulation of PSY expression does not necessarily result in an increase of the carotenoid content. Only those light conditions which allow chlorophyll biosynthesis lead to a significant increase of the carotenoid content. Therefore, it is proposed that up-regulation of PSY mRNA levels leads to an increased capacity for the formation of carotenoids. However, this only takes place under light conditions leading to protochlorophyllide photoconversion.

Alkyl and Aryl Transferases↗
PubMed1996

Phytoene synthase from Narcissus pseudonarcissus: functional expression, galactolipid requirement, topological distribution in chromoplasts and induction during flowering.

A cDNA coding for the carotenoid biosynthetic enzyme phytoene synthase was cloned from a Narcissus pseudonarcissus flower cDNA library, and the corresponding protein was overexpressed in insect cells using the baculovirus lipofection system. The full-length overexpressed enzyme exhibited very reduced catalytic activity compared with an overexpressed N-truncated form, with its transit sequence removed by site-directed mutagenesis. The shortened form readily bound quantitatively to lipid bilayers. Although it was active with liposomes prepared from plastid lipids, with phospholipid liposomes it was not, even though association took place. In this latter case, free galactose was capable of substituting for galactolipids, resulting in enzymatic activity. It is concluded that galactolipids are involved in catalytic activity, but do not serve as a membrane anchor. Antibodies raised against the recombinant enzyme made it possible to distinguish between a membrane-bound and a soluble, protein-complexed inactive form of phytoene synthase, present in the chromoplast stroma. These findings and data on phytoene synthase mRNA and protein expression presented here are discussed in terms of a possible regulatory role in color formation during chromoplast (flower) development.

Alkyl and Aryl Transferases↗
PubMed1994

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli.

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, beta-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for beta-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.

Alkyl and Aryl Transferases↗
PubMed1993

SC-0051, a 2-benzoyl-cyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase.

Growth inhibition of Lemna gibba plantlets by the bleaching herbicide, SC-0051 (2-(2-chloro-4-methanesulfonylbenzoyl)-1,3-cyclohexanedione)) was alleviated by the addition of homogentisic acid to the growth medium. Homogentisic acid is a key intermediate in the biosynthesis of tyrosine-derived plant quinones as well as in tyrosine metabolism. The herbicide prevented the incorporation of radioactivity from [14C]tyrosine into lipophilic plant metabolites and, in rat liver extracts, the herbicide inhibited the conversion of tyrosine to homogentisic acid. The enzyme p-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) from both Zea mays seedlings and liver tissues, was found to be subject to strong inhibition by SC-0051. Inhibition of plant quinone biosynthesis is a new mode of herbicidal action. One of the consequences of quinone depletion in plants by SC-0051. Inhibition of plant quinone biosynthesis is a new mode of herbicidal action. One of the consequences of quinone depletion in plants in vivo is apparently an indirect inhibition of phytoene desaturation. The enzyme phytoene desaturase itself, however, is not afflicted by the herbicide.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1993

In vitro expression and activity of lycopene cyclase and beta-carotene hydroxylase from Erwinia herbicola.

The cyclisation of lycopene to beta-carotene and the hydroxylation of beta-carotene to zeaxanthin are common enzymatic steps in the biosynthesis of carotenoids in a wide range of bacteria, fungi, and plants. We have individually expressed in E. coli the two genes coding for these enzymatic steps in Erwinia herbicola. The cyclase and hydroxylase enzymes have apparent molecular weights of 43 kDa and 22 kDa, respectively, as determined by SDS-PAGE. Hydroxylase in vitro activity was obtained only in the cytoplasmic fraction. Cyclase also demonstrated enzyme activity in a crude cell-free lysate, although to a lesser extent.

Base Sequence↗
PubMed1991

Distinct lipid compositions of parasite and host cell plasma membranes from Plasmodium chabaudi-infected erythrocytes.

Mouse erythrocytes infected with early or late trophozoites of the malaria parasite Plasmodium chabaudi were fractionated into free parasites and host cell plasma membranes, and both fractions were analyzed for cholesterol content and the composition of phospholipids and total fatty acids. The major results are: (i) parasites contain only a very low level of cholesterol which is about one-tenth of that of host cell plasma membranes. (ii) Parasites also contain less sphingomyelin and phosphatidylserine as well as more phosphatidylcholine than host cell plasma membranes. (iii) Parasites contain less 18:0 and 18:1 and more 18:2 and 20:4 fatty acids than host cell plasma membranes. (iv) During intraerythrocytic growth of parasites from early to late trophozoites, the relative proportions of cholesterol and phospholipids remain largely unchanged in both parasites and host cell plasma membranes. However, significant changes occur in the fatty composition of both compartments. There is an increase in the 20:4 and a decrease in the 18:0 and 18:1 fatty acids. (v) Plasma membranes of infected and non-infected erythrocytes exhibit about the same cholesterol content and phospholipid composition, but differ in the total fatty acid composition. Our data suggest the existence of distinct mechanisms controlling the different lipid compositions of parasites and host cell plasma membranes in whole Plasmodium chabaudi-infected erythrocytes during intraerythrocytic development of parasites, though both compartments are known to depend on the supply of various lipids from the host.

Animals↗
PubMed1991

Carotenoids of Erwinia herbicola and an Escherichia coli HB101 strain carrying the Erwinia herbicola carotenoid gene cluster.

Carotenoid pigments of Erwinia herbicola and a transformed strain of Escherichia coli carrying the carotenoid biosynthesis gene cluster of E. herbicola have been analyzed. Both organisms are capable of making essentially the same carotenoids, indicating that all of the genes required for the biosynthesis of the wild type E. herbicola carotenoids have been transformed intact into E. coli. The major products in both species of bacteria are beta-cryptoxanthin glucoside, zeaxanthin monoglucoside and zeaxanthin diglucoside. These compounds are the first example of secondary, non-allylic carotenoid glucosides. The absolute configuration 3R,3'R for zeaxanthin diglucoside was determined from its circular dichroism spectrum. Both species of bacteria also accumulate small amounts of hydrocarbon carotenes with similar cis/trans isomerization states.

Carotenoids↗
PubMed1990

Quinone compounds are able to replace molecular oxygen as terminal electron acceptor in phytoene desaturation in chromoplasts of Narcissus pseudonarcissus L.

The desaturation of phytoene to zeta-carotene and of zeta-carotene to lycopene employs molecular oxygen as the terminal electron acceptor. 2,3,5,6-Tetramethyl-1,4-benzoquinone (duroquinone) and other artificial quinones are able to replace oxygen, which demonstrates that oxygen does not act in a mixed-function oxygenase-like mechanism at the desaturase itself, but at a spatially separate site. Evidence for additional redox elements mediating between desaturase and oxygen is presented.

Carotenoids↗
PubMed1990

Chlorophyll-free chromoplasts from daffodil contain most of the enzymes for chlorophyll synthesis in a highly active form.

Chromoplasts isolated from chlorophyll-free daffodil flowers utilize in vitro delta-aminolevulinic acid (ALA) as precursor for the synthesis of large amounts of at least nine different products. Their identification as intermediates of the chlorophyll biosynthetic pathway demonstrates the presence of the majority of the respective enzymes in this nongreen plastid preparation. Porphobilinogen synthase was investigated more closely and found to be similar in its properties to the corresponding enzyme from other plastid sources. Protoporphyrin IX was also accepted as a substrate by chromoplast homogenate; here, as in the case of ALA as a substrate, Mg-protoporphyrin IX monomethyl ester was the last product formed. Formation of the isocyclic chlorophyll ring was not observed.

Aminolevulinic Acid↗
PubMed1989

Molecular oxygen and the state of geometric isomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts.

The membrane-bound carotenogenic reaction sequence in daffodil chromoplasts can be subdivided in vitro into three reaction segments by varying the incubation parameters O2 and light. In the first segment, 15-cis-phytoene is desaturated to 15-cis-zeta-carotene (trans elimination of hydrogen) in the dark and in the presence of O2 as an essential cofactor. A photoisomerization of the 15-cis double bond of the accumulated zeta-carotene to trans is the prerequisite for the function of the second segment, the desaturation to 7,9,9',7'-tetra-cis-lycopene (prolycopene, cis elimination of hydrogen). The role of O2 as an electron acceptor is discussed and evidence for an oxidoreductase acting as a redox mediator between the desaturase (forming the polyene chromophore) and O2 is presented. A certain analogy to the desaturation of stearoyl-carrier protein, which also occurs in plastids, is proposed. The third segment, the cyclization of prolycopene, is active only in the absence of O2 and involves additional cis-trans isomerization reactions.

Carbon Radioisotopes↗
PubMed1988

Biosynthesis of monoterpene hydrocarbons by isolated chromoplasts from daffodil flowers.

Incubation studies using [1-14C]isopentenyl diphosphate and [1-2H2]isopentenyl diphosphate as substrates revealed that isolated chromoplasts from flowers of Narcissus pseudonarcissus L. are able to synthesize monoterpene hydrocarbons and linalool in high yields. The enzymes involved are soluble in the chromoplast stroma. It is hypothesized that in the plant cell plastids are the site of monoterpene biosynthesis, whereas the formation of sesquiterpenes may be restricted to the cytoplasm/endoplasmic reticulum.

Carbon Radioisotopes↗
PubMed1985

Solubilization and reconstitution of the membrane-bound carotenogenic enzymes from daffodil chromoplasts.

The membrane-bound carotenogenic enzymes of daffodil (Narcissus pseudonarcissus) chromoplast membranes, i.e. dehydrogenation, cis-trans isomerization and cyclization activities, were solubilized in an enzymatically inactive form using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylamino]-1-propanesulfonate (Chaps). Full enzymatic activities were regained upon reconstitution of the solubilized proteins into liposomes. These preparations converted radiolabelled cis-phytoene into beta-carotene in a high yield. The reconstituted enzymatic sequence behaved as a tight 'assembly line'. In the enzymatic sequence of the reconstituted system the cis-trans isomerization reaction occurred on the stage of cis-phytofluene after a dehydrogenation of cis-phytoene.

Biotransformation↗