Measurement of Serine Acetyltransferase Activity in Crude Plant Extracts by a Coupled Assay System Using Cysteine Synthase

Nakamura, Katsuhito; Hayama, Atsushi; Morimoto, Masahiro; Fukushima, Kazuo; Tamura, Goro

doi:10.1093/oxfordjournals.pcp.a077370

Cited by 63 publications

(39 citation statements)

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“…4A) and the absence of CS activity (data not shown). The recombinant EhSAT showed an apparent K m of 0.22 Ϯ 0.05 mM for acetyl-CoA and 0.41 Ϯ 0.09 mM for L-serine, comparable with those reported for the bacterial (1) and plant SATs (3)(4)(5). Double reciprocal plots in the presence or absence of 3 or 10 M L-cysteine showed that the EhSAT activity was inhibited by L-cysteine in a competitive manner with L-serine but not with acetyl-CoA (Fig.…”

Section: Cloning Of Ehsat Andsupporting

confidence: 85%

“…Biochemical studies using purified (1)(2)(3)(4) and recombinant enzymes (5), as well as a genetic approach using a yeast two-hybrid system, revealed that CS and SAT form a heteromeric complex. SAT activity and O-acetylserine availability are the major regulatory factors in the control of the L-cysteine production in plants (6,7).…”

Section: Ec 23130) This Final Reaction Forming L-cysteine By Tramentioning

confidence: 99%

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Characterization of the Gene Encoding Serine Acetyltransferase, a Regulated Enzyme of Cysteine Biosynthesis from the Protist ParasitesEntamoeba histolyticaand Entamoeba dispar

Nozaki

Asai

Sánchez

et al. 1999

Journal of Biological Chemistry

120

113

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The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36 -52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by Lcystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.The cysteine biosynthetic pathway plays an important role in incorporation of inorganic sulfur into organic compounds. In bacteria and plants, L-cysteine is the precursor of most sulfurcontaining metabolites including methionine and glutathione. Extracellular sulfate is first imported by specific transporters. Intracellular sulfate is then activated by ATP sulfurylase and adenosine-5Ј-phosphosulfate kinase to form adenosine-5Ј-phosphosulfate and 3Ј-phosphoadenosine 5Ј-phosphosulfate, respectively. These activated sulfates are further reduced to sulfide. Sulfide then reacts with O-acetylserine, which is produced from serine and acetyl-CoA by serine acetyltransferase (SAT, 1 EC 2.3.1.30). This final reaction forming L-cysteine, by transfer of the alanyl moiety of O-acetylserine to sulfide, is catalyzed by L-cysteine synthase (CS; O-acetyl-L-serine (thiol)-lyase, EC 4.2.99.8). In contrast to bacteria and plants, animals are presumed to lack the sulfur assimilation pathway and thus require exogenous methionine as a sulfur source. Biochemical studies using purified (1-4) and recombinant enzymes (5), as well as a genetic approach using a yeast two-hybrid system, revealed that CS and SAT form a heteromeric complex. SAT activity and O-acetylserine availability are the major regulatory factors in the control of the L-cysteine production in plants (6, 7). Cytosolic isoforms of the SAT from Citrullus vulgaris and A...

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Section: Cloning Of Ehsat Andsupporting

confidence: 85%

Section: Ec 23130) This Final Reaction Forming L-cysteine By Tramentioning

confidence: 99%

Characterization of the Gene Encoding Serine Acetyltransferase, a Regulated Enzyme of Cysteine Biosynthesis from the Protist ParasitesEntamoeba histolyticaand Entamoeba dispar

Nozaki

Asai

Sánchez

et al. 1999

Journal of Biological Chemistry

120

113

View full text Add to dashboard Cite

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“…The enzymatic activity of SiR was measured in a total volume of 0.1 mL, containing 25 mM HEPES, pH 7.8, 1 mM Na 2 SO 3 , 5 mM OAS, 1 mg of recombinant OAS-TL C (Wirtz et al, 2004), 10 mM DTT, 30 mM NaHCO 3 , 15 mM Na 2 S 2 O 4 , and 5 mM methyl viologen along with the crude leaf extract. SAT activity was assayed by coupling to the OAS-TL reaction (Nakamura et al, 1987). To ensure high excess of OAS-TL activity during coupling of both reactions, all SAT activity determinations were supplemented with 4 units of purified recombinant OAS-TL.…”

Section: Determination Of Enzymatic Activities and Immunological Detementioning

confidence: 99%

Sulfite Reductase Defines a Newly Discovered Bottleneck for Assimilatory Sulfate Reduction and Is Essential for Growth and Development in Arabidopsis thaliana

et al. 2010

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The role of sulfite reductase (SiR) in assimilatory reduction of inorganic sulfate to sulfide has long been regarded as insignificant for control of flux in this pathway. Two independent Arabidopsis thaliana T-DNA insertion lines (sir1-1 and sir1-2), each with an insertion in the promoter region of SiR, were isolated. sir1-2 seedlings had 14% SiR transcript levels compared with the wild type and were early seedling lethal. sir1-1 seedlings had 44% SiR transcript levels and were viable but strongly retarded in growth. In mature leaves of sir1-1 plants, the levels of SiR transcript, protein, and enzymatic activity ranged between 17 and 28% compared with the wild type. The 28-fold decrease of incorporation of 35 S label into Cys, glutathione, and protein in sir1-1 showed that the decreased activity of SiR generated a severe bottleneck in the assimilatory sulfate reduction pathway. Root sulfate uptake was strongly enhanced, and steady state levels of most of the sulfur-related metabolites, as well as the expression of many primary metabolism genes, were changed in leaves of sir1-1. Hexose and starch contents were decreased, while free amino acids increased. Inorganic carbon, nitrogen, and sulfur composition was also severely altered, demonstrating strong perturbations in metabolism that differed markedly from known sulfate deficiency responses. The results support that SiR is the only gene with this function in the Arabidopsis genome, that optimal activity of SiR is essential for normal growth, and that its downregulation causes severe adaptive reactions of primary and secondary metabolism.

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“…Proteins were quantified as described by Bradford (1976) using bovine serum albumin as a standard. The enzymatic activities of SAT and OAS-TL were determined according to Nakamura et al (1987): SAT activity was assayed by coupling to the OAS-TL reaction. All SAT activity determinations were supplemented with 2 units of purified recombinant OAS-TL (Wirtz et al, 2004) to ensure high excess of OAS-TL activity during coupling of both reactions.…”

Section: Determination Of Enzyme Activitiesmentioning

confidence: 99%

Mitochondrial Serine Acetyltransferase Functions as a Pacemaker of Cysteine Synthesis in Plant Cells

et al. 2008

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(R.Q., A.B.) Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide. Thus, Cys synthesis in plants is significantly different from that in nonphotosynthetic eukaryotes at the cellular level.

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Measurement of Serine Acetyltransferase Activity in Crude Plant Extracts by a Coupled Assay System Using Cysteine Synthase

Cited by 63 publications

References 0 publications

Characterization of the Gene Encoding Serine Acetyltransferase, a Regulated Enzyme of Cysteine Biosynthesis from the Protist ParasitesEntamoeba histolyticaand Entamoeba dispar

Characterization of the Gene Encoding Serine Acetyltransferase, a Regulated Enzyme of Cysteine Biosynthesis from the Protist ParasitesEntamoeba histolyticaand Entamoeba dispar

Sulfite Reductase Defines a Newly Discovered Bottleneck for Assimilatory Sulfate Reduction and Is Essential for Growth and Development in Arabidopsis thaliana

Mitochondrial Serine Acetyltransferase Functions as a Pacemaker of Cysteine Synthesis in Plant Cells

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