Identification and characterization of an Arabidopsis homogentisate phytyltransferase paralog

Tocochromanols (tocopherols and tocotrienols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. There is considerable interest in identifying the genes involved in tocochromanol biosynthesis to allow transgenic modification of both tocochromanol levels and tocochromanol composition in agricultural crops. The first committed reaction in tocopherol biosynthesis is the condensation of homogentisic acid (HGA) with phytyldiphosphate or geranylgeranyldiphosphate, catalyzed by the homogentisate phytyltransferase (VTE2) or by the homogentisate geranylgeranyl transferase (HGGT). In this study, we describe the identification of conserved amino acid sequences within VTE2 and HGGT and the application of these conserved sequences for a motif analysis resulting in the discovery of a VTE2-paralog in the Arabidopsis genome. We designated this new gene VTE2-2 and renamed the old VTE2 to VTE2-1. Seed-specific expression of VTE2-2 in Arabidopsis resulted in increased seed-tocopherol levels, similar to the transgenic expression of VTE2-1. Bioinformatics analysis revealed that VTE2-2 is conserved in both monocotyledonous and dicotyledonous plants and is distinct from VTE2-1 and HGGT.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Tyamagondlu V. Venkatesh, and Balasulojini Karunanandaa have equally contributed.     

The <Emphasis Type="Italic">pds2</Emphasis> mutation is a lesion in the <Emphasis Type="Italic">Arabidopsis</Emphasis> homogentisate solanesyltransferase gene involved in plastoquinone biosynthesis

Plastoquinone plays critical roles in photosynthesis, chlororespiration and carotenoid biosynthesis. The previously isolated pds2 mutant from Arabidopsis was deficient in tocopherol and plastoquinone accumulation, and the biochemical phenotype of this mutant could not be reversed by externally applied homogentisate, suggesting a later step in tocopherol and/or plastoquinone biosynthesis had been disrupted. Recently, the protein encoded by At3g11950 (AtHST) was shown to condense homogentisate with solanesyl diphosphate (SDP), the substrate for plastoquinone synthesis, but not phytyl diphosphate (PDP), the substrate for tocopherol biosynthesis. We have sequenced the AtHST allele in the pds2 mutant background and identified an in-frame 6 bp (2 aa) deletion in the gene. The pds2 mutation could be functionally complemented by constitutive expression of AtHST, demonstrating that the molecular basis for the pds2 mutation is this 6 bp-lesion in the AtHST gene. Confocal microscopy of EGFP tagged AtHST suggested that AtHST is localized to the chloroplast envelope, supporting the hypothesis that plastoquinone synthesis occurs in the plastid.     

L-Tyrosine oxidative metabolism byStreptomyces aureofaciens

Summary L-Tyrosine metabolism found in normal mammalian system via homogentisic acid was investigated inStreptomyces aureofaciens S-834 as L-tyrosine was shown to possess the stimulatory role in the production of chlortetracycline antibiotic. L-Tyrosine as well as the substrates of the intermediary oxidative enzymes-p-hydroxyphenylpyruvate (p-HPPA) hydroxylase and homogentisate (HGA) oxygenase were incubated with the washed, resting cells of the culture and by adding sodium diethyldithiocarbamate and,-dipyridyl, the specific inhibitors of these enzymes to the culture, the oxidative intermediates-p-HPPA and HGA were blocked from further utilisation and were accumulated and isolated by extraction with the appropriate solvent system. The intermediates consisted of keto acids and aromatic phenolic acids were separated by adsorption and elution on silica gel and sephadex gel columns. p-HPPA and acetoacetate (AA) were identified as keto acids by obtaining their 2,4-dinitrophenylhydrazone derivatives. HGA as well as p-HPPA were identified as phenolic acids along with HGA lactone using diazotized reagents. These investigations helped to establish L-tyrosine oxidative pathway inStreptomyces aureofaciens via p-HPPA, HGA and AA leading to the biosynthesis of chlortetracycline antibiotic.     

Synthesis of plastoquinone-9 and phytylplastoquinone from homogentisate in lettuce chloroplasts

Chloroform-soluble extracts of unpurified chloroplast preparations of lettuce, pea and spinach and of class I lettuce chloroplasts that have been incubated in the light with [methylene-³H]homogentisate contain ³H-labelled plastoquinones-9 and -8 (minor homologue), 2-demethylplastoquinones-9 and -8 (minor homologue), pytylplastoquinone and 2-demethylphytylplastoquinone.. The absence of demethylquinols, the presumed precursors of the dimethylquinones, from the extracts to the fact that no precautions were taken in the extraction procedure to present their oxidation to the corresponding quinones.In unpurified lettuce chloroplasts the synthesis of these compounds from [methylene-³H]homogentisate is Mg²⁺-dependent and it is stimulated by light. The addition of isopentenyl pyrophosphate to the incubation mixtures increases the amounts of both groups of quinones (polyprenyl quinones and phytyl quinones) synthesised in the light and the amounts of polyprenyl quinones synthesised in the dark. Replacement of isopentenyl pyrophosphate with a source of preformed polyrenyl pyrophosphates brings about a marked rise in the amounts of polyprenyl quinones synthesized. This rise in polyprenyl quinone synthesis is further increased if the chloroplats are subjects to osmotic shock. The presence of S-adenosylmethionine increases the amounts of dimethylquinones synthesized at the expense of the demethylquinones. The implied precursor-product relationships between 2-demethylphytylplastoquinone (quinol?) and phytylplastoquinone and between the 2-demethylplastoquinones (quinols) and plastoquinones were verified in a pulse-labelling experiment. Confirmation that these quinones, or their corresponding quinols, are synthesized.     

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

Homogentisate is the precursor in the biosynthesis of -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 Homogentisate 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.     

Catalytic Reactions of the Homogentisate Prenyl Transferase Involved in Plastoquinone-9 Biosynthesis

Homogentisate solanesyl transferase (HST) catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl-1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. In vitro, HST from Spinacia oleracea L., Arabidopsis thaliana, and Chlamydomonas reinhardtii were all found to use not only solanesyl diphosphate but also short chain prenyl diphosphates of 10–20 carbon atoms as prenyl donors. Surprisingly, with these donors, prenyl transfer was largely decoupled from decarboxylation, and thus the major products were 6-prenyl-1,4-benzoquinol-2-methylcarboxylates rather than the expected 2-methyl-6-prenyl-1,4-benzoquinols. The 6-prenyl-1,4-benzoquinol-2-methylcarboxylates were not substrates for HST-catalyzed decarboxylation, and the enzyme kinetics associated with forming these products appeared quite distinct from those for 2-methyl-6-prenyl-1,4-benzoquinol formation in respect of catalytic rate, substrate K_m value, and the pattern of inhibition by haloxydine, a molecule that appeared to act as a dead end mimic of homogentisate. These observations were reconciled into a simple model for the HST mechanism. Here, prenyl diphosphate binds to HST to form at least two alternative complexes that go on to react differently with homogentisate and prenylate it either with or without it first being decarboxylated. It is supposed that solanesyl diphosphate binds tightly and preferentially in the mode that compels prenylation with decarboxylation.     

Accumulation of vitamin E in potato (Solanum tuberosum) tubers

Vitamin E (tocopherol) is a powerful antioxidant essential for human health and synthesized only by photosynthetic organisms. The effects of over-expression of tocopherol biosynthetic enzymes have been studied in leaves and seeds, but not in a non-photosynthetic, below-ground plant organ. Genetic and molecular approaches were used to determine if increased levels of tocopherols can be accumulated in potato (Solanum tuberosum L.) tubers through metabolic engineering. Two transgenes were constitutively over-expressed in potato: Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana homogentisate phytyltransferase (At-HPT). α-Tocopherol levels in the transgenic plants were determined by high-performance liquid chromatography. In potato tubers, over-expression of At-HPPD resulted in a maximum 266% increase in α-tocopherol, and over-expression of At-HPT yielded a 106% increase. However, tubers from transgenic plants still accumulated approximately 10- and 100-fold less α-tocopherol than leaves or seeds, respectively. The results indicate that physiological and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato tubers. Studying regulation and induction of tocopherol biosynthesis should reveal approaches to more effectively engineer crops with enhanced tocopherol content.     

Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm

Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and β-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4^Δ6,9,12,15)-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3^Δ9,12,15 or GLA, 18:3^Δ6,9,12), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.     

Increase of the tocochromanol content in transgenic Brassica napus seeds by overexpression of key enzymes involved in prenylquinone biosynthesis

Lipid soluble tocochromanols, only synthesised in photosynthetic organisms, are industrially interesting compounds because of their antioxidative properties and their essential function in nutrition. In order to increase the tocochromanol content in the seed oil of transgenic plants, approaches were undertaken to engineer the flux of substrates and intermediates through the pathway. To this end, we overexpressed genes encoding hydroxyphenylpyruvate dioxygenases, alone or in combination with chimeric homogentisate phytyltransferase and tocopherol cyclase genes, in seeds of transgenic Brassica napus plants and analysed total tocochromanol content and composition. Overexpression of chimeric hydroxyphenylpyruvate dioxygenase genes, both in the cytosol or in the plastids of developing seeds, yielded a slight although significant increase in total tocochromanol level. Coexpression of a hydroxyphenylpyruvate dioxygenase gene with both a homogentisate phytyltransferase gene and a tocopherol cyclase gene elevated this effect with maximum increases of up to two-fold in individual lines and this phenotype was found to be stably inherited. These data showed that the three enzymes are critical in determining the total tocochromanol content in the seed oil of Brassica napus plants, while the tocopherol cyclase, unlike hydroxyphenylpyruvate dioxygenase and homogentisate phytyltransferase, had additionally an effect on the relative abundance of individual tocochromanol species and resulted in an increase of δ-tocopherol and plastochromanol-8 in the seeds.Mirela Raclaru and Jens Gruber contributed equally