Cloning and analysis of the gene for cystathionine ?-synthase from Arabidopsis thaliana

Kim, Jung‐Sup; Leustek, Thomas

doi:10.1007/bf00041395

Cited by 67 publications

(35 citation statements)

References 35 publications

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“…2), showing that both possess CGS activity, confirming that the N-terminal domain of the mature plant CGS is not essential for its catalytic activity. These results extend the data of a previous report showing that deletion of part of the plantspecific N-terminal region of the mature Arabidopsis CGS enables complementation of an E. coli metB mutant (Kim and Leustek, 1996).…”

Section: Plant Cgss Contained An Extended N-terminal Region In Comparsupporting

confidence: 89%

The N-Terminal Region of Arabidopsis Cystathionine gamma -Synthase Plays an Important Regulatory Role in Methionine Metabolism

Hacham

2002

Plant Physiology

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Cystathionine ␥-synthase (CGS) is a key enzyme of Met biosynthesis in bacteria and plants. Aligning the amino acid sequences revealed that the plant enzyme has an extended N-terminal region that is not found in the bacterial enzyme. However, this region is not essential for the catalytic activity of this enzyme, as deduced from the complementation test of an Escherichia coli CGS mutant. To determine the function of this N-terminal region, we overexpressed full-length Arabidopsis CGS and its truncated version that lacks the N-terminal region in transgenic tobacco (Nicotiana tabacum) plants. Transgenic plants expressing both types of CGS had a significant higher level of Met, S-methyl-Met, and Met content in their proteins. However, although plants expressing full-length CGS showed the same phenotype and developmental pattern as wild-type plants, those expressing the truncated CGS showed a severely abnormal phenotype. These abnormal plants also emitted high levels of Met catabolic products, dimethyl sulfide and carbon disulfide. The level of ethylene, the Met-derived hormone, was 40 times higher than in wild-type plants. Since the alien CGS was expressed at comparable levels in both types of transgenic plants, we further suggest that post-translational modification(s) occurs in this N-terminal region, which regulate CGS and/or Met metabolism. More specifically, since the absence of the N-terminal region leads to an impaired Met metabolism, the results further suggest that this region plays a role in protecting plants from a high level of Met catabolic products such as ethylene.The sulfur-containing amino acid Met is an important essential amino acid in animal nutrition. Apart from its role as a protein constituent and its central function in initiating mRNA translation, Met indirectly regulates a variety of cellular processes as the precursor of S-adenosyl-Met (SAM), the primary biological methyl group donor. SAM is also the precursor of plant metabolites such as ethylene, polyamines, vitamin B1, and the Fe-chelator mugineic acid (Anderson, 1990;Ma et al., 1995;Sun, 1998). In addition, Met also serves as a donor for secondary metabolites through S-methyl-Met (SMM; Mudd and Datko, 1990). As can be expected of its cellular importance, Met biosynthesis is subject to complex regulatory control whose mechanism is only now being gradually clarified. Two main elements of this complex regulation have recently been elucidated in plants. In the first, the Met level is controlled by competition between its first specific enzyme, cystathionine ␥-synthase (CGS), and Thr synthase, for their common substrate, O-phosphohomo-Ser. Evidence of this competition and its role in Met synthesis was recently obtained by analyzing a mto2-1 mutant of Arabidopsis. This mutant, in which the gene encoding Thr synthase is impaired, demonstrated a approximately 22-fold higher accumulation of soluble Met in rosette leaves than wild-type Arabidopsis (Bartlem et al., 2000).A second regulatory mechanism of Met synthesis in plants occurs at the level...

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Section: Plant Cgss Contained An Extended N-terminal Region In Comparsupporting

confidence: 89%

The N-Terminal Region of Arabidopsis Cystathionine gamma -Synthase Plays an Important Regulatory Role in Methionine Metabolism

Hacham

2002

Plant Physiology

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“…Molecular cloning, comparison to bacterial enzymes, and in vitro assays of enzyme activity have confirmed that the A. thaliana At3g01120 gene encodes cystathionine γ-synthase (EC 2.5.1.48; Figure 7), the committing enzyme for methionine biosynthesis (Kim and Leustek, 1996;Ravanel et al, 1998). Another A. thaliana locus, At1g33320, has 76% amino acid sequence identity to At3g01120, but there is as yet no confirmation that this gene encodes a cystathionine γ-synthase.…”

Section: Methionine Biosynthesismentioning

confidence: 99%

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

Jander

Joshi

2009

The Arabidopsis Book

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The aspartate-derived amino acid pathway in plants leads to the biosynthesis of lysine, methionine, threonine, and isoleucine. These four amino acids are essential in the diets of humans and other animals, but are present in growthlimiting quantities in some of the world's major food crops. Genetic and biochemical approaches have been used for the functional analysis of almost all Arabidopsis thaliana enzymes involved in aspartate-derived amino acid biosynthesis. The branch-point enzymes aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase, cystathionine gamma synthase, threonine synthase, and threonine deaminase contain well-studied sites for allosteric regulation by pathway products and other plant metabolites. In contrast, relatively little is known about the transcriptional regulation of amino acid biosynthesis and the mechanisms that are used to balance aspartatederived amino acid biosynthesis with other plant metabolic needs. The aspartate-derived amino acid pathway provides excellent examples of basic research conducted with A. thaliana that has been used to improve the nutritional quality of crop plants, in particular to increase the accumulation of lysine in maize and methionine in potatoes.

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“…Since alliinase is supposed to be a vacuolar protein [3], it needs a signal peptide [15]; ASA, A. sativum alliinase [15]; ATCBL, Arabidopsis thaliana cystathionine β-lyase [41]; CGS-1, A. thaliana cystathionine γ-synthase [42]; CYS-3, S. cerevisiae cystathionine γ-lyase [43]; MET17, S. cerevisiae cystathionine γ-synthase [45]; METC, S. cerevisiae cystathionine β-lyase [46]; METZ, Pseudomonas aeruginosa O-succinyl homoserine sulfhydrylase [47]; MGL, P. putida methionine γ-lyase [48].…”

Section: Molecular Comparison With Alliinases From Other Plantsmentioning

confidence: 99%

Alliinase [S‐alk(en)yl‐ L‐cysteine sulfoxide lyase] from Allium tuberosum (Chinese chive)

Manabe

Hasumi

Sugiyama

et al. 1998

European Journal of Biochemistry

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Alliinase [S-alk(en)yl-L-cysteine sulfoxide lyase], a pyridoxal-phosphate-(Pxy-P)-dependent enzyme, is responsible for the degradative conversion of S-alk(en)yl-L-cysteine sulfoxide to volatile odorous sulfurcontaining metabolites in Allium plants. We have purified alliinase from shoots of Allium tuberosum (Chinese chive) to apparent homogeneity by SDS/polyacrylamide gel electrophoresis. A cDNA clone encoding alliinase was isolated from a cDNA library constructed from whole plants of A. tuberosum by hybridization screening with a synthetic 50-residue oligonucleotide encoding a conserved region of the alliinases from onion and garlic. The isolated cDNA encoded a protein of 476 amino acid residues with a molecular mass of 54 083 Da. The deduced amino acid sequence exhibited 66Ϫ69% identities with those of reported alliinases from onion, garlic and shallot. The partial amino acid sequence, which was determined for a V8 protease-digested peptide fragment of the purified alliinase, was perfectly matched with the sequence deduced from the cDNA. An expression vector of recombinant alliinase cDNA was constructed in yeast. The catalytically active protein was in the soluble fraction of transformed yeast. Site-directed mutagenesis experiments indicated that Lys280 was essential for the catalytic activity and, thus, a possible Pxy-P-binding residue. The mRNA expression of the alliinase gene comprising a multigene family in the shoots of green plants was twofold higher than that in the roots of green plants ; however, the expression in the shoots of etiolated plants was only 13% that in green shoots, although the expression in the roots was not remarkably different between in green and etiolated plants. Immunohistochemical investigation indicated that the alliinase protein is predominantly accumulated in the bundle sheath cells of shoots of A. tuberosum.Keywords : Allium; alliinase ; cDNA cloning; pyridoxal 5′-phosphate; sulfur metabolism.The plants belonging to genus Allium generally produce characteristic odorous sulfur-containing compounds. These volatile sulfur compounds are formed from non-volatile S-alk(en)yl-L-cysteine sulfoxide (ACS), as a precursor, through enzymatic degradation, which is catalyzed by so-called alliinase [S-alk-(en)yl-L-cysteine sulfoxide lyase] [1, 2]. In onion (Allium cepa) cells, alliinase is assumed to be localized in vacuoles, thereby being separated from ACS present in the cytosol [3]. Upon rupture of the cells, the enzyme can react with the substrate to yield sulfenic acid as the first sulfur-containing volatile compound (Scheme 1). Numerous sulfur-containing metabolites, such as thiosulfinates, thiols, disulfides, etc. are secondarily produced from sulfenic acid through non-enzymatic reactions [4]. These

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Cloning and analysis of the gene for cystathionine ?-synthase from Arabidopsis thaliana

Cited by 67 publications

References 35 publications

The N-Terminal Region of Arabidopsis Cystathionine gamma -Synthase Plays an Important Regulatory Role in Methionine Metabolism

The N-Terminal Region of Arabidopsis Cystathionine gamma -Synthase Plays an Important Regulatory Role in Methionine Metabolism

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

Alliinase [S‐alk(en)yl‐ L‐cysteine sulfoxide lyase] from Allium tuberosum (Chinese chive)

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