Arabidopsis Enzyme Research Articles

Primary N-assimilation into Amino Acids in Arabidopsis

The assimilation of inorganic nitrogen into organic form has marked effects on plant productivity, biomass, and crop yield (Hageman and Lambert, 1988; Lawlor et al., 1989). As such, a tremendous amount of biochemical and physiological studies have been performed on nitrogen assimilatory enzymes from a variety of plant species, especially crops and legumes. Summaries of such biochemical studies can be found in several comprehensive reviews (Miflin and Lea, 1976, 1977; Miflin, 1980; Miflin and Lea, 1980; Miflin and Lea, 1980, 1982; Miflin and Cullimore, 1984; Poulton et al., 1989). Although these biochemical studies have provided a solid groundwork, a complete picture of the N-assimilation process and its regulation in a single plant is still lacking for a number of reasons. The existence of multiple isoenzymes for each step in nitrogen assimilation has confounded biochemical purification as have the technical difficulties of isoenzyme purification and organelle isolation. As the mechanisms controlling intraand intercellular transport of inorganic and organic nitrogen in plants are still under investigation, it is impossible to predict the in vivo function of nitrogen assimilatory enzymes localized in distinct cells or subcellular compartments based on in vitro biochemistry. Plant mutants have provided a mechanism to dissect the process of N-assimilation in vivo (Lea and Forde, 1994) (Lam et al., 1996). The aim of this chapter is to specifically highlight and update examples where molecular, genetic, and biochemical analyses of Nassimilation genes and mutants in Arabidopsis have begun to define the in vivo roles of individual isoenzymes in plant nitrogen assimilation and to uncover the mechanisms regulating this process. In all higher plants, inorganic nitrogen is first reduced to ammonia prior to its incorporation into organic form (Lea, 1993). For a review of the regulation of Nitrate assimilation and reduction in Arabidopsis see (Crawford and Forde, 2002). Ammonia is assimilated into organic form as glutamine and glutamate, which serve as the nitrogen donors in the biosynthesis of essentially all amino acids, nucleic acids, and other nitrogen-containing compounds such as chlorophyll (Lea, 1993). The individual isoenzymes of GS, GOGAT or GDH have been proposed to play roles in three major ammonium assimilation processes: (i) primary nitrogen assimilation; (ii) reassimilation of photorespiratory ammonia; and (iii) reassimilation of recycled nitrogen. For a review of these processes see (Stewart et al., 1980; Lea, 1993). Glutamine and glutamate can then be used to form aspartate and asparagine, and these four amino acids are used to translocate organic nitrogen from sources to sinks (Lea and Miflin, 1980; Peoples and Gifford, 1993). The enzymes involved in the primary assimilation of ammonium into these four N-transport amino acids Glu/Gln and Asp/Asn are shown in Fig. 1 and include; glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT) and asparagine synthetase (AS). While most studies of nitrogen metabolism have previously been performed in legumes and crop species, HPLC analyses of Arabidopsis has demonstrated that these same four amino acids can account for 60-64% of the total free amino acids present in Arabidopsis leaves and are also transported in the vascular tissues (Fig. 2) (Schultz, 1994; Lam et al., 1995). Thus, Arabidopsis appears to be a suitable model plant for the study of nitrogen assimilation into primary amino acids and the results should have impact on less genetically tractable crop plants. It is noteworthy that each enzyme for GS, GOGAT, AspAT and AS exists as multiple isoenzymes, encoded by multiple genes, even in Arabidopsis (Fig. 1). Table 1 lists the enzymes, the genes, and available mutants for these N-assimilatory enzymes in Arabidopsis. Below, we review how studies of the genes and mutants for these isoenzymes in Arabidopsis have helped to illuminate the role of specific genes in this Nassimilation pathway.

Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases.

Sweet basil (Ocimum basilicum) peltate glandular trichomes produce a variety of small molecular weight phenylpropanoids, such as eugenol, caffeic acid, and rosmarinic acid, that result from meta hydroxylation reactions. Some basil lines do not synthesize eugenol but instead synthesize chavicol, a phenylpropanoid that does not contain a meta hydroxyl group. Two distinct acyltransferases, p-coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase and p-coumaroyl-coenzyme A:4-hydroxyphenyllactic acid p-coumaroyl transferase, responsible for the production of p-coumaroyl shikimate and of p-coumaroyl 4-hydroxyphenyllactate, respectively, were partially purified and shown to be specific for their substrates. p-Coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase is expressed in basil peltate glands that are actively producing eugenol and is not active in glands of noneugenol-producing basil plants, suggesting that the levels of this activity determine the levels of synthesis of some meta-hydroxylated phenylpropanoids in these glands such as eugenol. Two basil cDNAs encoding isozymes of cytochrome P450 CYP98A13, which meta hydroxylates p-coumaroyl shikimate, were isolated and found to be highly similar (90% identity) to the Arabidopsis homolog, CYP98A3. Like the Arabidopsis enzyme, the basil enzymes were found to be very specific for p-coumaroyl shikimate. Finally, additional hydroxylase activities were identified in basil peltate glands that convert p-coumaroyl 4-hydroxyphenyllactic acid to its caffeoyl derivative and p-coumaric acid to caffeic acid.

Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants

5-Formyltetrahydrofolate cycloligase (5-FCL) catalyzes the conversion of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) to 5,10-methenyltetrahydrofolate and is considered to be the main means whereby 5-CHO-H(4)PteGlu(n) is metabolized in mammals, yeast, and bacteria. 5-CHO-H(4)PteGlu(n) is known to occur in plants and to be highly abundant in leaf mitochondria. Genomics-based approaches identified Arabidopsis and tomato cDNAs encoding proteins homologous to 5-FCLs of other organisms but containing N-terminal extensions with the features of mitochondrial targeting peptides. These homologs were shown to have 5-FCL activity by characterizing recombinant enzymes produced in Escherichia coli and by functional complementation of a yeast fau1 mutation with the Arabidopsis 5-FCL cDNA. The recombinant Arabidopsis enzyme is active as a monomer, prefers the penta- to the monoglutamyl form of 5-CHO-H(4)PteGlu(n), and has kinetic properties broadly similar to those of 5-FCLs from other organisms. Enzyme assays and immunoblot analyses indicated that 5-FCL is located predominantly if not exclusively in plant mitochondria and that the mature, active enzyme lacks the putative targeting sequence. Serine hydroxymethyltransferase (SHMT) from plant mitochondria was shown to be inhibited by 5-CHO-H(4)PteGlu(n) as are SHMTs from other organisms. Since mitochondrial SHMT is crucial to photorespiration, 5-FCL may help prevent 5-CHO-H(4)PteGlu(n) from reaching levels that would inhibit this process. Consistent with this possibility, 5-FCL activity was far higher in leaf mitochondria than root mitochondria.

α-Dioxygenase, a new enzyme in fatty acid metabolism

Infection of tobacco leaves with bacteria resulted in the accumulation of a 75-kDa oxygenase. This enzyme and a homologous one from Arabidopsis were expressed in insect cells and their catalytic properties were studied. Linolenic acid incubated with preparations containing the recombinant enzymes was converted into 2( R)-hydroperoxylinolenic acid, an unstable oxylipin which underwent spontaneous degradation into a mixture of 8( Z), 11( Z), 14( Z)-heptadecatrienal, 2( R)-hydroxylinolenic acid, and 8( Z), 11( Z), 14( Z)-heptadecatrienoic acid. Studies with stereospecifically deuterated linolenic acid demonstrated that the enzymatic α-dioxygenation proceeded with retention of absolute configuration. The α-dioxygenases from tobacco and Arabidopsis showed homology to mammalian prostaglandin endoperoxide synthases-1 and -2 and to linoleate diol synthase from certain fungi. His-388 and His-207 (heme-binding), Gln-203 (charge-stabilizing), and Tyr-385 (hydrogen-abstracting) of ovine endoperoxide synthase-1 were conserved in the α-dioxygenases, and mutation studies of the Arabidopsis enzyme showed that their replacement with other amino acids resulted in loss of catalytic activity. The α-dioxygenase activity in tobacco leaves increased from three- to fourfold upon mechanical wounding, about fourfold in response to infection by Pseudomonas syringae pv syringae (incompatible strain), and from six- to eightfold in response to P. syringae pv tabaci (compatible strain).

An Arabidopsis gene encoding an alpha-xylosyltransferase involved in xyloglucan biosynthesis.

Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of alpha-xylosyltransferase and beta-glucan synthase activities. Here we report that etiolated pea microsomes contain an alpha-xylosyltransferase that catalyzes the transfer of xylose from UDP-[(14)C]xylose onto beta(1,4)-linked glucan chains. The solubilized enzyme had the capacity to transfer xylosyl residues onto cello-oligosaccharides having 5 or more glucose residues. Analysis of the data from these biochemical assays led to the identification of a group of Arabidopsis genes and the hypothesis that one or more members of this group may encode alpha-xylosyltransferases involved in xyloglucan biosynthesis. To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured with the biochemical assay described above. One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity. Characterization of the radiolabeled products obtained with cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the (14)C-labeled xylose mainly to the second glucose residue from the nonreducing end. Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an alpha(1,6)-linkage to the glucan chain. We conclude that this Arabidopsis gene encodes an alpha-xylosyltransferase activity involved in xyloglucan biosynthesis.

Guard cell- and phloem idioblast-specific expression of thioglucoside glucohydrolase 1 (myrosinase) in Arabidopsis.

Thioglucoside glucohydrolase 1 (TGG1) is one of two known functional myrosinase enzymes in Arabidopsis. The enzyme catalyzes the hydrolysis of glucosinolates into compounds that are toxic to various microbes and herbivores. Transgenic Arabidopsis plants carrying beta-glucuronidase and green fluorescent protein reporter genes fused to 0.5 or 2.5 kb of the TGG1 promoter region were used to study spatial promoter activity. Promoter activity was found to be highly specific and restricted to guard cells and distinct cells of the phloem. No promoter activity was detected in the root or seed. All guard cells show promoter activity. Positive phloem cells are distributed in a discontinuous pattern and occur more frequent in young tissues. Immunocytochemical localization of myrosinase in transverse and longitudinal sections of embedded material show that the TGG1 promoter activity reflects the position of the myrosinase enzyme. In the flower stalk, the myrosinase-containing phloem cells are located between phloem sieve elements and glucosinolate-rich S cells. Our results suggest a cellular separation of myrosinase enzyme and glucosinolate substrate, and that myrosinase is contained in distinct cells. We discuss the potential advantages of locating defense and communication systems to only a few specific cell types.

Cloning and Characterization of an S-Formylglutathione Hydrolase from Arabidopsis thaliana

A cDNA from Arabidopsis thaliana resembling S-formylglutathione hydrolase (SFGH), an enzyme with putative roles in formaldehyde detoxification in animals and microorganisms, has been cloned and expressed in Escherichia coli. The purified recombinant Arabidopsis enzyme (AtSFGH) was a dimer composed of 31-kDa subunits. Like SFGHs from other sources, AtSFGH had thioesterase activity toward S-formylglutathione and carboxyesterase activity toward 4-methylumbelliferyl acetate. Unlike other SFGHs, the enzyme from Arabidopsis actively hydrolyzed S-acetylglutathione. AtSFGH activity was inhibited by heavy metals and sulfhydryl alkylating agents, but was insensitive to serine hydrolase inhibitors, suggesting that the enzyme was a cysteine-dependent hydrolase. Although Atsfgh transcripts were determined in plants and cultures of Arabidopsis, the respective enzyme could not be detected in planta after the esterase activities present were resolved using isoelectric focusing. Instead, Arabidopsis contained several carboxyesterases active toward α-naphthyl acetate, which were all sensitive to inhibition by the serine hydrolase inhibitor paraoxon.

Over-expression of cystathionine γ-synthase in Arabidopsis thaliana leads to increased levels of methionine and S-methylmethionine

Cystathionine γ-synthase (CGS, EC 4.2.99.9), the first committed enzyme in methionine biosynthesis, was over-expressed in Arabidopsis thaliana by introducing in the genome of this plant the coding sequence of the Arabidopsis enzyme under the control of the cauliflower mosaic virus 35S promoter. In order to target the recombinant protein to the chloroplast, the transgene included the sequence encoding the N-terminal transit peptide of Arabidopsis CGS. CGS activity and polypeptide were increased several fold in these plants. There was a markedly increased level of soluble methionine in the leaves of the transformed plants, up to 15-fold, indicating that CGS is a rate-limiting enzyme in this metabolic pathway. In addition, the transformed plants strongly over-accumulated S-methylmethionine, but not S-adenosylmethionine, in agreement with the view that S-methylmethionine corresponds to a storage form of labile methyl groups in plants and/or plays a role in preventing S-adenosylmethionine accumulation. The same strategy was used to increase the level of cystathionine β-lyase (CBL, EC 4.4.1.8), the second committed enzyme in methionine biosynthesis, in transformed A. thaliana. Despite an increase in both CBL activity and polypeptide in transformed Arabidopsis plants over-expressing Arabidopsis CBL, there was very little change in the contents of soluble methionine and S-methylmethionine, suggesting strongly that CBL is not rate limiting in the methionine biosynthetic pathway.

HUMAN AND PLANT FLAVIN-CONTAINING MONOOXYGENASE N-OXYGENATION OF AMINES: DETOXICATION VS. BIOACTIVATION

Generally, the flavin-containing monooxygenase of mammalian systems has been considered a detoxication enzyme converting nucleophilic heteroatom-containing chemicals into polar, more readily excreted metabolites. The beneficial effects of this process are thought to be participation in the detoxication of foodstuffs and other xenobiotics that might otherwise be bioactivated by other enzyme systems. The physiological role of mammalian FMO is unknown although it has been observed that many heteroatom-containing chemicals in plants are efficiently oxygenated by FMO. Recently, a plant FMO-like enzyme has been characterized and it appears to possess many of the same functional properties of mammalian FMO. The advantage of studying the FMO-like genes and enzymes in Arabidopsis as a model system allows for the investigation of the role of FMO-like enzymes in plant growth and development. The Arabidopsis FMO-like gene product, while resembling the mammalian counterpart in many respects, is quite distinct. However, study of the Arabidopsis FMO-like enzyme may provide considerable insight into the possible role of mammalian FMOs in biogenic amine metabolism.

N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana.

The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase)/fructose-2,6-bisphosphatase (F26BPase) catalyses the formation and degradation of the regulatory metabolite fructose 2,6-bisphosphate. A cDNA encoding the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric properties of the affinity-purified enzyme were characterized. In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and P(i), several metabolites were identified as important new effectors. PP(i), phosphoenolpyruvate and 2-phosphoglycerate strongly inhibited F6P,2K activity, whereas fructose 1,6-bisphosphate and 6-phosphogluconate inhibited F26BPase activity. Furthermore, pyruvate was an activator of F6P,2K and an inhibitor of F26BPase. Both kinase and phosphatase activities were rapidly inactivated by mild heat treatment (42 degrees C, 10 min), but the presence of phosphate protected both enzyme activities from inactivation. In addition to the catalytic regions, the Arabidopsis enzyme comprises a 345-amino-acid N-terminus of unknown function. The role of this region was examined by the expression of a series of N-terminally truncated enzymes. The full-length and truncated enzymes were analysed by gel-filtration chromatography. The full-length enzyme was eluted as a homotetramer, whereas the truncated enzymes were eluted as monomers. Deletion of the N-terminus decreased the kinase/phosphatase activity ratio by 4-fold, and decreased the affinity for the substrate fructose 6-phosphate. The data show that the N-terminus is important both for subunit assembly and for defining the kinetic properties of the enzyme.

N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase)/fructose-2,6-bisphosphatase(F26BPase) catalyses the formation and degradation of the regulatory metabolite fructose 2,6-bisphosphate. A cDNA encoding the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric properties of the affinity-purified enzyme were characterized. In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and Pi, several metabolites were identified as important new effectors. PPi, phosphoenolpyruvate and 2-phosphoglycerate strongly inhibited F6P,2K activity, whereas fructose 1,6-bisphosphate and 6-phosphogluconate inhibited F26BPase activity. Furthermore, pyruvate was an activator of F6P,2K and an inhibitor of F26BPase. Both kinase and phosphatase activities were rapidly inactivated by mild heat treatment (42°C, 10min), but the presence of phosphate protected both enzyme activities from inactivation. In addition to the catalytic regions, the Arabidopsis enzyme comprises a 345-amino-acid N-terminus of unknown function. The role of this region was examined by the expression of a series of N-terminally truncated enzymes. The full-length and truncated enzymes were analysed by gel-filtration chromatography. The full-length enzyme was eluted as a homotetramer, whereas the truncated enzymes were eluted as monomers. Deletion of the N-terminus decreased the kinase/phosphatase activity ratio by 4-fold, and decreased the affinity for the substrate fructose 6-phosphate. The data show that the N-terminus is important both for subunit assembly and for defining the kinetic properties of the enzyme.

Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis.

Arabidopsis (ecotype Columbia-0) genes, AtDEF1 and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase. Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences. Radiolabeled full-length AtDEF1 was imported and processed by isolated pea (Pisum sativum L. Laxton's Progress No. 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts. The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed in Escherichia coli and purified using C-terminal hexahistidyl tags. Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E. coli enzyme. Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively. Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development. The results suggest an essential role for peptide deformylase in protein processing in all plant plastids.

Molecular characterization and regulation of formate dehydrogenase inArabidopsis thaliana

In a previous publication we reported the purification of formate dehydrogenase (FDH, EC 1.2.1.2.) from Arabidopsis thaliana (L.) Heyn. and some of its properties. Here we report the identification of an Arabidopsis FDH cDNA, as well as studies of the molecular characterization and regulation of the enzyme in Arabidopsis. FDH is present as a single-copy gene in the Arabidopsis genome and is located on chromosome V. The deduced amino acid sequence of the Arabidopsis enzyme shows over 80% identity with those from other plants (potato, barley, rice). Northern and western blots show that the FDH mRNA and protein levels in Arabidopsis leaves are similar to those in flowers and stems and higher than those in roots. The effects of chemical and environmental factors on FDH expression in leaves were investigated and compared with their effects on formaldehyde dehydrogenase (FADH, EC 1.2.1.1.). Induction of FDH by one-carbon metabolites, such as methanol, formaldehyde, and formate, was observed at the protein level, but changes at the transcript level were small and different from those observed for FADH. While the steady-state levels of FDH transcripts increased quickly (within hours) and strongly in response to various stresses, protein amounts increased slowly, after 1 to 3 days; FADH transcripts showed no observable change. The possible mechanism of regulation of FDH at both the transcript and protein levels is discussed.Key words: Arabidopsis, formate dehydrogenase, formate metabolism, one-carbon metabolites, stress.

Distinct patterns of expression but similar biochemical properties of protein L-isoaspartyl methyltransferase in higher plants.

Protein L-isoaspartyl methyltransferase is a widely distributed repair enzyme that initiates the conversion of abnormal L-isoaspartyl residues to their normal L-aspartyl forms. Here we show that this activity is expressed in developing corn (Zea mays) and carrot (Daucus carota var. Danvers Half Long) plants in patterns distinct from those previously seen in winter wheat (Triticum aestivum cv Augusta) and thale cress (Arabidopsis thaliana), whereas the pattern of expression observed in rice (Oryza sativa) is similar to that of winter wheat. Although high levels of activity are found in the seeds of all of these plants, relatively high levels of activity in vegetative tissues are only found in corn and carrot. The activity in leaves was found to decrease with aging, an unexpected finding given the postulated role of this enzyme in repairing age-damaged proteins. In contrast with the situation in wheat and Arabidopsis, we found that osmotic or salt stress could increase the methyltransferase activity in newly germinated seeds (but not in seeds or seedlings), whereas abscisic acid had no effect. We found that the corn, rice, and carrot enzymes have comparable affinity for methyl-accepting substrates and similar optimal temperatures for activity of 45 degrees C to 55 degrees C as the wheat and Arabidopsis enzymes. These experiments suggest that this enzyme may have specific roles in different plant tissues despite a common catalytic function.

Antisense inhibition of the beta-carotene hydroxylase enzyme in Arabidopsis and the implications for carotenoid accumulation, photoprotection and antenna assembly.

The xanthophylls are oxygenated carotenoids and are important structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex apoproteins (LHC) and contribute to photoprotection via non-photochemical quenching of chlorophyll fluorescence (NPQ) in oxygenic photosynthetic organisms. Previously, mutations have been described that disrupt many steps in the xanthophyll biosynthetic pathway. However, there are no definitive reports of a lesion that effects the beta-hydroxylase enzyme, which catalyzes hydroxylation of the beta-rings of beta-carotene and alpha-carotene, and is thus necessary for synthesis of essentially all xanthophylls of higher plant chloroplasts. We have utilized an antisense approach to effectively reduce levels of beta-hydroxylase in Arabidopsis thaliana in order to examine how a reduction in this enzyme impacts carotenoid biosynthesis and plant viability. Expression of the antisense beta-hydroxylase transgene resulted in a maximal reduction in violaxanthin of 64% and a maximal reduction in neoxanthin of 41%. This reduction was reflected in a 22% increase in beta-carotene and a reduction in the total carotenoid pool, whereas lutein levels were relatively unaltered. Despite the reduction in violaxanthin and neoxanthin, the antisense beta-hydroxylase plants had a wild-type complement of chlorophylls and LHCs on a fresh weight basis. Under high light stress, the unconverted pool of violaxanthin was the same size as in wild type and thus there was an even greater proportional reduction in zeaxanthin of 75%. Despite this marked decrease in zeaxanthin, NPQ only declined by 16%.

In Vivo Phosphorylation of Arabidopsis thaliana 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase

Fructose 2,6-bisphosphate (F2,6BP) is a non-metabolic sugar-phosphate ubiquitously found in eukaryotes. This compound regulates the balance of glycolysis and gluconeogenesis. In plants, its physiological functions are thought to be an inhibition of sucrose synthesis in source leaves and to be an activation of glycolysis in sink organs. Both synthesis and degradation of F2,6BP are catalyzed by a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase (PFK 2/FBPase 2; EC 2.7.1.105/EC 3.1.3.46). In animals and yeast, it is well known that several isozymes are regulated by protein phosphorylation. In plant enzymes, on the other hand, it had been thought that the protein phosphorylation is not involved in the enzymatic regulation. An Arabidopsis cDNA for PFK 2/FBPase 2 was isolated and expressed in E. coli. Antisera prepared from the recombinant enzyme identified 96-kD and 92-kD protein bands of plant crude protein extracts. Results of in vitro calf intestine alkaline phosphatase treatment and an immunoprecipitation of the crude extracts from [32P]-labeled whole plants revealed that the 96-kD protein was phosphorylated in vivo. Immunoblot analysis showed that the 96-kD protein was enriched in younger leaves than in older leaves. These results indicate that Arabidopsis enzyme is phosphorylated. Now we try to identify the biochemical effects of this phosphorylation, especially whether this phosphorylation can change the enzymatic activities or not.

The catabolic function of the alpha-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine-oxoglutarate reductase, but not saccharopine dehydrogenase.

Whereas plants and animals use the alpha-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine-oxoglutarate reductase (lysine-ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the alpha-amino adipic acid pathway in plants.

The catabolic function of the α-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine–oxoglutarate reductase, but not saccharopine dehydrogenase

Whereas plants and animals use the α-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine–oxoglutarate reductase (lysine–ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the α-amino adipic acid pathway in plants.

Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes

Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis database as encoding putative cytidine deaminases. Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds to theArabidopsis thaliana gene,cda1. The deduced amino acid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes. The cDNA shares 44% amino acid identity with theEscherichia coli cytidine deaminase but only 26 and 27% identity with human and yeast enzymes. A unique zinc-binding domain of the Ecoli enzyme forms the active site. A similar putative zinc-binding domain was identified in the Arabidopsis enzyme based upon primary sequence similarities. These similarities permitted us to model the active site of the Arabidopsis enzyme upon that of the Ecoli enzyme. In this model, the active site zinc is coordinated by His73, Cys103, Cys107, and an active site hydroxyl. Additional residues that participate in catalysis, Asn64, Glu66, Ala78, Glu79, and Pro102, are conserved between the Arabidopsis and Ecoli enzymes suggesting that the Arabidopsis enzyme has a catalytic mechanism similar to the Ecoli enzyme. The two overlapping ESTs were used to prepare a single, full-length clone corresponding to theA thaliana cda1 cDNA. This cDNA was subcloned into pProExHtb and expressed as a fusion protein with an N-terminal His6 tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes both cytidine (Km = 226 µand 2’-deoxycytidine (Km= 49 µM) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP.

Identification and expression of a cDNA for human glycolate oxidase

The isolation and expression of a human liver cDNA encoding a 43 kDa protein with glycolate oxidase activity is described. The cDNA (HAO1) is 1128 bp and has a 1113 bp open reading frame. Northern blot analysis detects a 1.8 kb mRNA with expression restricted to the liver. The 370 amino acid protein has 89% sequence similarity to glycolate oxidase from mouse liver and 53% similarity to the spinach and Arabidopsis enzymes. The protein has glycolate oxidase activity in vitro, as measured by the reduction of 2,6-dichlorophenolindophenol in the presence of flavin mononucleotide and glycolate. The genomic sequence is contained within eight exons and encompasses approximately 57 kb of chromosome 20p12. This paper offers the first full functional description of a gene encoding human glycolate oxidase.