Regulation of Serine Biosynthesis in Arabidopsis

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In plants, Ser is biosynthesized by two different pathways: a photorespiratory pathway via Gly and a plastidic pathway via the phosphorylated metabolites from 3-phosphoglycerate. In contrast to the better characterization of the photorespiratory pathway at a molecular level, the molecular regulation and significance of the plastidic pathway are not yet well understood. An Arabidopsis thaliana cDNA encoding 3-phosphoserine phosphatase, the enzyme that is responsible for the con-

mentioning

confidence: 74%

Plastidic Pathway of Serine Biosynthesis

Noji

Saito

1999

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“…Despite this non-conservative substitution, the secondary structures of McyI, NdaH, and PGDH did not appear to differ at this site, with each protein possessing a helix-loop-sheet composition. The fact that A. thaliana PGDH contains glycine in place of Arg 62 , as do most of the other cyanobacterial dehydrogenases, suggests that certain substitutions at this position may not be critical to activity because the A. thaliana PGDH can complement E. coli PGDH (serA) mutants (29 hypothesis. As Trp 139 also plays a key role in the cooperativity of Ser binding and inhibition, we predict that McyI is a noncooperative enzyme.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

Pearson

Barrow

Neilan

2007

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thiotemplate function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of D-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce ␣-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.The hepatotoxic microcystins compose the largest and most structurally diverse group of cyanobacterial toxins. Over 65 isoforms of microcystin varying by degree of methylation, hydroxylation, epimerization, peptide sequence, and toxicity have been identified (1). Underlying the extraordinary heterogeneity present among the microcystins is their common cyclic structure and possession of several rare nonproteinogenic amino acid moieties. Collectively, the microcystins may be described as monocyclic heptapeptides containing both D-and L-amino acids plus N-methyldehydroalanine and a unique ␤-amino acid side group, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Fig. 1) (2). Although various amino acid substitutions can occur within the microcystin cyclic structure, the most common toxin isoform, microcystin LR, contains lysine and arginine at positions 2 and 4, respectively (Fig. 1).The microcystin biosynthesis (mcy) gene cluster was the first complex metabolite gene cluster to be fully sequenced from a cyanobacterium. In Microcystis aeruginosa PCC7806, the mcy gene cluster spans 55 kb and comprises 10 genes arranged in two divergently transcribed operons, mcyA-C and mcyD-J. Although most open reading frames within the mcy gene cluster have been assigned a function based on homologous entries in the data bases, no obvious function could be assigned to the 1014-bp gene mcyI. Preliminary sequence analysis of the inferred primary peptide seq...

“…Enzyme Assay-PGDH activity was assayed as previously described with some modifications (22). Enzymatic activity of PGDH in the direction of NADH oxidation (the reverse reaction) was carried out at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…Arabidopsis plants were transformed using the floral dip method (28). Transformed plants were selected on Murashige and Skoog (MS) (29) agar media with 50 g/ml kanamycin and 25 g/ml To construct transgenic Arabidopsis plants expressing ApPGDH, which is targeted into the chloroplast, the fragment encoding a 60-amino acid transit peptide for chloroplast targeting was amplified from the Arabidopsis PGDH gene (22) by PCR using the primer set, preAtPGDH-NcoF and preAtPGDHNcoR. The amplified fragment was ligated into the NcoI site of pApPGDH-His6-SKϩ to generate the pAtTP-ApPGDH-His6-SKϩ vector.…”

Section: Methodsmentioning

confidence: 99%

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Metabolic Engineering for Betaine Accumulation in Microbes and Plants

Waditee

Bhuiyan

Hirata

et al. 2007

Plants accumulate a variety of osmoprotectants that improve their ability to combat abiotic stresses. Among them, betaine appears to play an important role in conferring resistance to stresses. Betaine is synthesized via either choline oxidation or glycine methylation. An increased betaine level in transgenic plants is one of the potential strategies to generate stress-tolerant crop plants. Here, we showed that an exogenous supply of serine or glycine to a halotolerant cyanobacterium Aphanothece halophytica, which synthesizes betaine from glycine by a three-step methylation, elevated intracellular accumulation of betaine under salt stress. The gene encoding 3-phosphoglycerate dehydrogenase (PGDH), which catalyzes the first step of the phosphorylated pathway of serine biosynthesis, was isolated from A. halophytica. Expression of the Aphanothece PGDH gene in Escherichia coli caused an increase in levels of betaine as well as glycine and serine. Expression of the Aphanothece PGDH gene in Arabidopsis plants, in which the betaine synthetic pathway was introduced via glycine methylation, further increased betaine levels and improved the stress tolerance. These results demonstrate that PGDH enhances the levels of betaine by providing the precursor serine for both choline oxidation and glycine methylation pathways.Salinity is a major problem affecting world overall agricultural production. Approximately 20% of the cultivated land worldwide is impaired by high salinity which causes ion imbalance and hyperosmotic stress in plants (1). Plants have evolved various strategies to cope with salinity, including the accumulation of low molecular weight organic compatible solutes (osmoprotectants) such as sugars, some amino acids, and quaternary ammonium compounds (2-4). Glycine betaine (N,N,N-trimethylglycine, hereafter betaine) is a major osmoprotectant to protect plants from high salinity (2-4). Most known biosynthetic pathways of betaine include a two-step oxidation of choline: choline 3 betaine aldehyde 3 betaine. The first step is catalyzed by choline monooxygenase (CMO) in plants (5), choline dehydrogenase (CDH) in animals and bacteria (6, 7), and choline oxidase in some bacteria (8, 9). The second step is catalyzed by betaine aldehyde dehydrogenase (BADH) in all organisms (6, 10, 11). Introducing the betaine synthetic pathway into betaine non-accumulating plants has been applied to improve salt tolerance of plants (8,(12)(13)(14)(15). However, the accumulation levels of betaine in the transformed plants are relatively low. Supplying betaine precursors such as choline, ethanolamine, and serine to the transformed plants enhance betaine accumulation. Thus, the availability of betaine precursors limits the betaine biosynthesis (15, 16 -18).Recently, we showed that a halotolerant cyanobacterium, Aphanothece halophytica (A. halophytica), synthesizes betaine from glycine by a three-step methylation, which is catalyzed by two N-methyltransferases (ApGSMT and ApDMT). ApGSMT is responsible for the two-step methylation reactions of ...