The tomato mutation nxd1 reveals a gene necessary for neoxanthin biosynthesis and demonstrates that violaxanthin is a sufficient precursor for abscisic acid biosynthesis.

Carotenoid pigments are indispensable for plant life. They are synthesized within plastids where they provide essential functions in photosynthesis. Carotenoids serve as precursors for the synthesis of the strigolactone phytohormones, which are made from ß-carotene, and of abscisic acid (ABA), which is produced from certain xanthophylls. Despite the significant progress that has been made in our understanding of the carotenoid biosynthesis pathway, the synthesis of the xanthophyll neoxanthin has remained unknown. We report here on the isolation of a tomato (Solanum lycopersicum) mutant, neoxanthin-deficient 1 (nxd1), which lacks neoxanthin, and on the cloning of a gene that is necessary for neoxanthin synthesis in both tomato and Arabidopsis. The locus nxd1 encodes a gene of unknown function that is conserved in all higher plants. The activity of NXD1 is essential but cannot solely support neoxanthin synthesis. Lack of neoxanthin does not significantly reduce the fitness of tomato plants in cultivated field conditions and does not impair the synthesis of ABA, suggesting that in tomato violaxanthin is a sufficient precursor for ABA production in vivo.

Elucidation of the pathway to astaxanthin in the flowers of Adonis aestivalis.

A few species in the genus Adonis are the only land plants known to produce the valuable red ketocarotenoid astaxanthin in abundance. Here, we ascertain the pathway that leads from the ß-rings of ß-carotene, a carotenoid ubiquitous in plants, to the 3-hydroxy-4-keto-ß-rings of astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione) in the blood-red flowers of Adonis aestivalis, an ornamental and medicinal plant commonly known as summer pheasant's eye. Two gene products were found to catalyze three distinct reactions, with the first and third reactions of the pathway catalyzed by the same enzyme. The pathway commences with the activation of the number 4 carbon of a ß-ring in a reaction catalyzed by a carotenoid ß-ring 4-dehydrogenase (CBFD), continues with the further dehydrogenation of this carbon to yield a carbonyl in a reaction catalyzed by a carotenoid 4-hydroxy-ß-ring 4-dehydrogenase, and concludes with the addition of an hydroxyl group at the number 3 carbon in a reaction catalyzed by the erstwhile CBFD enzyme. The A. aestivalis pathway is both portable and robust, functioning efficiently in a simple bacterial host. Our elucidation of the pathway to astaxanthin in A. aestivalis provides enabling technology for development of a biological production process and reveals the evolutionary origin of this unusual plant pathway, one unrelated to and distinctly different from those used by bacteria, green algae, and fungi to synthesize astaxanthin.

Inactivation of genes encoding plastoglobuli-like proteins in Synechocystis sp. PCC 6803 leads to a light-sensitive phenotype.

Plastoglobulins (PGL) are the predominant proteins of lipid globules in the plastids of flowering plants. Genes encoding proteins similar to plant PGL are also present in algae and cyanobacteria but in no other organisms, suggesting an important role for these proteins in oxygenic photosynthesis. To gain an understanding of the core and fundamental function of PGL, the two genes that encode PGL-like polypeptides in the cyanobacterium Synechocystis sp. PCC 6803 (pgl1 and pgl2) were inactivated individually and in combination. The resulting mutants were able to grow under photoautotrophic conditions, dividing at rates that were comparable to that of the wild-type (WT) under low-light (LL) conditions (10 microeinsteins x m(-2) x s(-1)) but lower than that of the WT under moderately high-irradiance (HL) conditions (150 microeinsteins x m(-2) x s(-1)). Under HL, each Deltapgl mutant had less chlorophyll, a lower photosystem I (PSI)/PSII ratio, more carotenoid per unit of chlorophyll, and very much more myxoxanthophyll (a carotenoid symptomatic of high light stress) per unit of chlorophyll than the WT. Large, heterogeneous inclusion bodies were observed in cells of mutants inactivated in pgl2 or both pgl2 and pgl1 under both LL and HL conditions. The mutant inactivated in both pgl genes was especially sensitive to the light environment, with alterations in pigmentation, heterogeneous inclusion bodies, and a lower PSI/PSII ratio than the WT even for cultures grown under LL conditions. The WT cultures grown under HL contained 2- to 3-fold more PGL1 and PGL2 per cell than cultures grown under LL conditions. These and other observations led us to conclude that the PGL-like polypeptides of Synechocystis play similar but not identical roles in some process relevant to the repair of photooxidative damage.

Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse beta-carotene 15, 15'-monooxygenase.

BACKGROUND: beta-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a beta-carotene-accumulating E. coli system. RESULTS: Our assays show that mutation of either Y235 or Y326 to leucine (no cation-pi stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our ab initio model of BCMO1 with beta-carotene mounted supports a mechanism involving cation-pi stabilization by Y235 and Y326. CONCLUSIONS: Our data are consistent with the formation of a substrate carbocation intermediate and cation-pi stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.

A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study.

Carotenoids are indispensable pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and are produced, as well, by many bacteria and fungi. Elucidation of biochemical pathways leading to the carotenoids that function in the photosynthetic membranes of land plants has been greatly aided by the use of carotenoid-accumulating strains of Escherichia coli as heterologous hosts for functional assays, in vivo, of the otherwise difficult to study membrane-associated pathway enzymes. This same experimental approach is uniquely well-suited to the discovery and characterization of yet-to-be identified enzymes that lead to carotenoids of the photosynthetic membranes in algal cells, to the multitude of carotenoids found in nongreen plant tissues, and to the myriad flavor and aroma compounds that are derived from carotenoids in plant tissues. A portfolio of plasmids suitable for the production in E. coli of a variety of carotenoids is presented herein. The use of these carotenoid-producing E. coli for the identification of cDNAs encoding enzymes of carotenoid and isoprenoid biosynthesis, for characterization of the enzymes these cDNAs encode, and for the production of specific carotenoids for use as enzyme substrates and reference standards, is described using the flowering plant Adonis aestivalis to provide examples. cDNAs encoding nine different A. aestivalis enzymes of carotenoid and isoprenoid synthesis were identified and the enzymatic activity of their products verified. Those cDNAs newly described include ones that encode phytoene synthase, beta-carotene hydroxylase, deoxyxylulose-5-phosphate synthase, isopentenyl diphosphate isomerase, and geranylgeranyl diphosphate synthase.

Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae.

Cyanidioschyzon merolae is considered to be one of the most primitive of eukaryotic photosynthetic organisms. To obtain insights into the origin and evolution of the pathway of carotenoid biosynthesis in eukaryotic plants, the carotenoid content of C. merolae was ascertained, genes encoding enzymes of carotenoid biosynthesis in this unicellular red alga were identified, and the activities of two candidate pathway enzymes of particular interest, lycopene cyclase and beta-carotene hydroxylase, were examined. C. merolae contains perhaps the simplest assortment of chlorophylls and carotenoids found in any eukaryotic photosynthetic organism: chlorophyll a, beta-carotene, and zeaxanthin. Carotenoids with epsilon-rings (e.g., lutein), found in many other red algae and in green algae and land plants, were not detected, and the lycopene cyclase of C. merolae quite specifically produced only beta-ringed carotenoids when provided with lycopene as the substrate in Escherichia coli. Lycopene beta-ring cyclases from several bacteria, cyanobacteria, and land plants also proved to be high-fidelity enzymes, whereas the structurally related epsilon-ring cyclases from several plant species were found to be less specific, yielding products with beta-rings as well as epsilon-rings. C. merolae lacks orthologs of genes that encode the two types of beta-carotene hydroxylase found in land plants, one a nonheme diiron oxygenase and the other a cytochrome P450. A C. merolae chloroplast gene specifies a polypeptide similar to members of a third class of beta-carotene hydroxylases, common in cyanobacteria, but this gene did not produce an active enzyme when expressed in E. coli. The identity of the C. merolae beta-carotene hydroxylase therefore remains uncertain.

Construction and characterization of genetically modified synechocystis sp. PCC 6803 photosystem II core complexes containing carotenoids with shorter pi-conjugation than beta-carotene.

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).

Key role of conserved histidines in recombinant mouse beta-carotene 15,15'-monooxygenase-1 activity.

Alignment of sequences of vertebrate beta-carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V(max). The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.

A study in scarlet: enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis.

The red ketocarotenoid astaxanthin (3,3'-dihydroxy-4,4'-diketo-beta,beta-carotene) is widely used as an additive in feed for the pigmentation of fish and crustaceans and is frequently included in human nutritional supplements as well. There is considerable interest in developing a plant-based biological production process for this valuable carotenoid. Adonis aestivalis (Ranunculaceae) is unusual among plants in synthesizing and accumulating large amounts of astaxanthin and other ketocarotenoids. The formation of astaxanthin requires only the addition of a carbonyl at the number 4 carbon of each beta-ring of zeaxanthin (3,3'-dihydroxy-beta,beta-carotene), a carotenoid typically present in the green tissues of higher plants. We screened an A. aestivalis flower library to identify cDNAs that might encode the enzyme that catalyzes the addition of the carbonyls. Two closely related cDNAs selected in this screen were found to specify polypeptides similar in sequence to plant beta-carotene 3-hydroxylases, enzymes that convert beta-carotene (beta,beta-carotene) into zeaxanthin. The Adonis enzymes, however, exhibited neither 4-ketolase nor 3-hydroxylase activity when presented with beta-carotene as the substrate in Escherichia coli. Instead, the products of the Adonis cDNAs were found to modify beta-rings in two distinctly different ways: desaturation at the 3,4 position and hydroxylation of the number 4 carbon. The 4-hydroxylated carotenoids formed in E. coli were slowly metabolized to yield compounds with ketocarotenoid-like absorption spectra. It is proposed that a 3,4-desaturation subsequent to 4-hydroxylation of the beta-ring leads to the formation of a 4-keto-beta-ring via an indirect and unexpected route: a keto-enol tautomerization.

Inactivation of sll1556 in Synechocystis strain PCC 6803 impairs isoprenoid biosynthesis from pentose phosphate cycle substrates in vitro.

In cyanobacteria many compounds, including chlorophylls, carotenoids, and hopanoids, are synthesized from the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate. Isoprenoid biosynthesis in extracts of the cyanobacterium Synechocystis strain PCC 6803 grown under photosynthetic conditions, stimulated by pentose phosphate cycle substrates, does not appear to require methylerythritol phosphate pathway intermediates. The sll1556 gene, distantly related to type 2 IPP isomerase genes, was disrupted by insertion of a Kanr cassette. The mutant was fully viable under photosynthetic conditions although impaired in the utilization of pentose phosphate cycle substrates. Compared to the parental strain the Deltasll1556 mutant (i) is deficient in isoprenoid biosynthesis in vitro with substrates including glyceraldehyde-3-phosphate, fructose-6-phosphate, and glucose-6-phosphate; (ii) has smaller cells (diameter ca. 13% less); (iii) has fewer thylakoids (ca. 30% less); and (iv) has a more extensive fibrous outer wall layer. Isoprenoid biosynthesis is restored with pentose phosphate cycle substrates plus the recombinant Sll1556 protein in the Deltasll1556 supernatant fraction. IPP isomerase activity could not be demonstrated for the purified Sll1556 protein under our in vitro conditions. The reduction of thylakoid area and the effect on outer wall layer components are consistent with an impairment of isoprenoid biosynthesis in the mutant, possibly via hopanoid biosynthesis. Our findings are consistent with an alternate metabolic shunt for biosynthesis of isoprenoids.

Isoprenoid biosynthesis in Synechocystis sp. strain PCC6803 is stimulated by compounds of the pentose phosphate cycle but not by pyruvate or deoxyxylulose-5-phosphate.

The photosynthetic cyanobacterium Synechocystis sp. strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-C-methyl-D-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in Escherichia coli, or by deoxyxylulose-5-phosphate, the first pathway intermediate in E. coli. However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds. Fosmidomycin (at 1 micro M and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect [(14)C]IPP incorporation stimulated by DHAP plus GA3P. To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium. The combined results suggest that the MEP pathway, as described for E. coli, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in Synechocystis sp. strain PCC6803. Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis.