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Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation. [ABSTRACT FROM AUTHOR]

Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase ( HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate ( GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase ( HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate ( PDP). An HGGT-independent pathway for tocotrienol synthesis has also been shown to occur by de-regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co-expression of Arabidopsis hydroxyphenylpyruvate dioxygenase ( HPPD) and Escherichia coli bi-functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2-1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA-expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP: PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA-induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT-catalyzed tocotrienol synthesis. [ABSTRACT FROM AUTHOR]

The biosynthesis of the tocotrienol and tocopherol forms of vitamin E is initiated by prenylation of homogentisate. Geranylgeranyl diphosphate (GGDP) is the prenyl donor for tocotrienol synthesis, whereas phytyl diphosphate (PDP) is the prenyl donor for tocopherol synthesis. We have previously shown that tocotrienol synthesis is initiated in monocot seeds by homogentisate geranylgeranyl transferase (HGGT). This enzyme is related to homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol synthesis. Here we show that monocot HGGT is localized in the plastid and expressed primarily in seed endosperm. Despite the close structural relationship of monocot HGGT and HPT, these enzymes were found to have distinct substrate specificities. Barley ( Hordeum vulgare cv. Morex) HGGT expressed in insect cells was six times more active with GGDP than with PDP, whereas the Arabidopsis HPT was nine times more active with PDP than with GGDP. However, only small differences were detected in the apparent K values of barley HGGT for GGDP and PDP. Consistent with its in vitro substrate properties, barley HGGT generated a mixture of tocotrienols and tocopherols when expressed in the vitamin E-null vte2-1 mutant lacking a functional HPT. Relative levels of tocotrienols and tocopherols produced in vte2-1 differed between organs and growth stages, reflective of the composition of plastidic pools of GGDP and PDP. In addition, HGGT was able to functionally substitute for HPT to rescue vte2-1-associated phenotypes, including reduced seed viability and increased fatty acid oxidation of seed lipids. Overall, we show that monocot HGGT is biochemically distinct from HPT, but can replace HPT in important vitamin E-related physiological processes. [ABSTRACT FROM AUTHOR]

Glutathione biosynthesis is a key component in the network of plant stress responses that counteract oxidative damage and maintain intracellular redox environment. Using a combination of mass spectrometry and site-directed mutagenesis, we examined the response of Arabidopsis thaliana glutamate-cysteine ligase (GCL) to changes in redox environment. Mass spectrometry identified two disulfide bonds (Cys186-Cys406 and Cys349-Cys364) in GCL. Mutation of either Cys-349 or Cys-364 to a Ser reduced reaction rate by twofold, but substitution of a Set for either Cys-186 or Cys-406 decreased activity by 20-fold and abrogated the response to changes in redox environment. Redox titrations show that the regulatory disulfide bond has a midpoint potential comparable with other known redox-responsive plant proteins. Mutation of Cys-102, Cys-251, Cys-349, or Cys-364 did not alter the response to redox environment, indicating that modulation of activity depends on the Cys186-Cys406 disulfide bond. In vivo analysis of GCL in Arabidopsis root extracts revealed that multiple oxidative stresses altered the distribution of oxidized (active) and reduced (inactive) enzyme and that this change correlated with increased GCL activity. The thiol-based regulation of GCL provides a posttranslational mechanism for modulating enzyme activity in response to in vivo redox environment and suggests a role for oxidative signaling in the maintenance of glutathione homeostasis in plants. [ABSTRACT FROM AUTHOR]

In plants, cysteine biosynthesis plays a central role in fixing inorganic sulfur from the environment and provides the only metabolic sulfide donor for the generation of methionine, glutathione, phytochelatins, iron-sulfur clusters, vitamin cofactors, and multiple secondary metabolites. O-Acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis, the pyridoxal 5′-phosphate (PLP)-dependent conversion of O-acetylserine into cysteine. Here we describe the 2.2 Å, resolution crystal structure of OASS from Arabidopsis thaliana (AtOASS) and the 2.7 Å resolution structure of the AtOASS K46A mutant with PLP and methionine covalently linked as an external aldimine in the active site. Although the plant and bacterial OASS share a conserved set of amino acids for PLP binding, the structure of AtOASS reveals a difference from the bacterial enzyme in the positioning of an active site loop formed by residues 74–78 when methionine is bound. Site-directed mutagenesis, kinetic analysis, and ligand binding titrations probed the functional roles of active site residues. These experiments indicate that Asn77 and Gin147 are key amino acids for O-acetylserine binding and that Thr74 and Ser75 are involved in sulfur incorporation into cysteine. In addition, examination of the AtOASS structure and nearly 300 plant and bacterial OASS sequences suggest that the highly conserved β8A-β9A surface loop may be important for interaction with serine acetyltransferase, the other enzyme in cysteine biosynthesis. Initial protein-protein interaction experiments using AtOASS mutants targeted to this loop support this hypothesis. [ABSTRACT FROM AUTHOR]

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of γ-glutamylcysteine from glutamate (Km = 9.1 ms) and cysteine (Km = 2.7 mM). Glutathione feedback inhibits AtGCL (Ki ∼1.0 nM). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random ter-reactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress. [ABSTRACT FROM AUTHOR]

Vitamin E tocochromanols are generated in plants by prenylation of homogentisate using geranylgeranyl diphosphate (GGDP) for tocotrienol biosynthesis and phytyl diphosphate (PDP) for tocopherol biosynthesis. Homogentisate geranylgeranyl transferase (HGGT), which uses GGDP for prenylation, is a proven target for oilseed tocochromanol biofortification that effectively bypasses the chlorophyll-linked pathway that limits PDP for vitamin E biosynthesis. In this report, we explored the feasibility of maximizing tocochromanol production in the oilseed crop camelina (Camelina sativa) by combining seed-specific HGGT expression with increased biosynthesis and/or reduced homogentisate catabolism. Plastid-targeted Escherichia coli TyrA -encoded chorismate mutase/prephenate dehydrogenase and Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) cDNA were co-expressed in seeds to bypass feedback-regulated steps and increase flux into homogentisate biosynthesis. Homogentisate catabolism was also suppressed by seed-specific RNAi of the gene for homogentisate oxygenase (HGO), which initiates homogentisate degradation. In the absence of HGGT expression, tocochromanols were increased by ∼2.5-fold with HPPD/TyrA co-expression, and ∼1.4-fold with HGO suppression compared to levels in non-transformed seeds. No further increase in tocochromanols was observed in HPPD/TyrA lines with the addition of HGO RNAi. HGGT expression alone increased tocochromanol concentrations in seeds by ∼four-fold to ≤1400 μg/g seed weight. When combined with HPPD/TyrA co-expression, we obtained an additional three-fold increase in tocochromanol concentrations indicating that homogentisate concentrations limit HGGT's capacity for maximal tocochromanol production. The addition of HGO RNAi further increased tocochromanol concentrations to 5000 μg/g seed weight, an unprecedented tocochromanol concentration in an engineered oilseed. Metabolomic data obtained from engineered seeds provide insights into phenotypic changes associated with "extreme" tocochromanol production. • Metabolic bypass combinations were explored to maximize oilseed vitamin E production. • HGGT overexpression plus enhanced homogentisate levels yielded high vitamin E titers. • Highest reported vitamin E tocochromanol concentrations in an oilseed were achieved. • Major seed metabolome alterations accompanied "extreme" vitamin E biosynthesis. [ABSTRACT FROM AUTHOR]

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para -coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para -coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para -coumarate, can bypass this blockage. Furthermore, a trans -cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans -cinnamate into para -coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para -coumarate for its subsequent β-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch. [ABSTRACT FROM AUTHOR]

In plants and Plasmodium falciparum, the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoethanolamine methyltransferase (PEAMT). This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and, on the basis of bioinformatics, was postulated to function in the nematode Caenorhabditis elegans. Here we describe the cloning and biochemical characterization of a PEAMT from C. elegans (gene, pmt- 2; protein, PMT-2). Although similar in size to the PEAMT from plants, which contain two tandem methyltransferase domains, PMT-2 retains only the C-terminal methyltransferase domain. RNA-mediated interference experiments in C. elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype. Unlike the plant and Plasmodium PEAMT, which catalyze all three methylations in the pathway, PMT-2 catalyzes only the last two steps in the pathway, i.e., the methylation of phosphomonomethylethanolamine (P-MME) to phosphodimethylethanolamine (P- DME) and of P-DME to phosphocholine. Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2. Product inhibition by S-adenosylhomocysteine was competitive versus S-adenosylmethionine and noncompetitive versus P-DME, consistent with formation of a dead-end complex. Inhibition by phosphocholine was competitive versus each substrate. Fluorescence titrations show that all substrates and products bind to the free enzyme. The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2. This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT. Our results indicate that nematodes also use the PEAMT pathway for phosphatidyicholine biosynthesis. If the essential role of PMT-2 in C. elegans is conserved in parasitic nematodes of mammals and plants, then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value. [ABSTRACT FROM AUTHOR]

The article focuses on engineering the biosynthesis of the plant polyketide resveratrol (trans-3,5,4'-trihydroxystilbene) in yeast and mammalian cells using unnatural protein fusions. The results show that unnatural fusion proteins offer a strategy for improving pathway yields by colocalizing 4-coumarate-CoA ligase and stilbene synthase. The engineering of a plant metabolic pathway in mammalian cells may give additional opportunities and applications for medical and pharmaceutical research.