Cystathionine γ-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli

Ravanel, Stéphane; Gakière, Bertrand; Job, Dominique; Douce, Roland

doi:10.1042/bj3310639

Cited by 89 publications

(114 citation statements)

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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: 98%

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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Section: Methionine Biosynthesismentioning

confidence: 98%

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

Jander

Joshi

2009

The Arabidopsis Book

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“…Met metabolite or protein can bind to this region, leading to conformational changes and inhibition of CGS catalytic activity. However, it should be taken into account that a study conducted with purified Arabidopsis CGS showed that metabolites such as Met, SAM, cystathionine, homo-Cys, SMM, S-adenosylhomo-Cys, 5-methylthioadenosine, Thr, and iso-Leu had no significant effect on CGS activity (Ravanel et al, 1998). At the same time, it is possible that some other metabolite or protein could interact with elements in this N-terminal region and alter CGS activity, such as Met catabolic products (ethylene or DMS, for example).…”

Section: Discussionmentioning

confidence: 99%

“…Plants expressing fulllength Arabidopsis CGS (lanes 2-4) exhibited two HA cross-reacting bands. The upper band migrated with the expected size of the natural mature Arabidopsis CGS (53 kD; Ravanel et al, 1998) plus the 3-kD HA tag. The second band migrated more rapidly, with an estimated size of 53 kD.…”

Section: Expression Of Arabidopsis Cgs In Transgenic Tobacco Plantsmentioning

confidence: 99%

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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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“…Based on determined K m values and estimated subcellular concentration of its substrates in Arabidopsis, it has been proposed that the CGS reaction normally proceeds at approximately 1-2% of its maximal rate, and therefore, CGS is not the rate-limiting step in methionine biosynthesis (Ravanel et al 1998a). Inhibitor studies of CGS in Lemna also revealed that only 16% of CGS control activity is necessary for normal rates of methionine biosynthesis and plant growth, and also that down-regulation of CGS to 15% of control activity is not sufficient to reduce the rate of methionine biosynthesis (Thompson et al 1982b).…”

Section: Threonine Biosynthesis Regulates Metabolic Flux To Methioninmentioning

confidence: 99%