Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.1.22 (cystathionine beta-synthase)
965 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the reaction connecting serine and cysteine/methionine metabolism, plays a regulatory role in cysteine biosynthesis in plants. We have isolated a cDNA clone encoding SATase by direct genetic complementation of a Cys- mutation in Escherichia coli using an expression library of Citrullus vulgaris (watermelon) cDNA. The cDNA encodes a polypeptide of 294 amino acids (31,536 Da) exhibiting 51% homology with that of E. coli SATase. DNA-blot analysis indicated the presence of a single copy of the SATase gene (sat) in watermelon. RNA hybridization analysis suggested the relatively ubiquitous and preferential expression in the hypocotyls of etiolated seedlings. Immunoblot analysis indicated the accumulation of SATase predominantly in etiolated plants. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the SATase in an allosteric manner, indicating the regulatory function of SATase in this metabolic pathway, whereas beta-(pyrazole-1-yl)-L-alanine, a secondary metabolite formed partly through the cysteine biosynthetic pathway, showed no inhibitory effect. A multi-enzyme complex was formed from recombinant proteins of SATase and cysteine synthase (O-acetylserine(thiol)-lyase) from watermelon, suggesting efficient metabolic channeling from serine to cysteine, preventing the diffusion of intermediary O-acetyl-L-serine.
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PMID:Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon. 760

Four new mutations in the cystathionine beta-synthase (CBS) gene have been identified in Italian patients with homocystinuria. The first mutation is a G-to-A transition at base 374 in exon 3, causing an arginine-to-glutamic acid substitution at position 125 of the protein (R125Q). This mutation has been found in homozygosity in a patient partially responsive to pyridoxine treatment. The second mutation is a C-to-T transition at base 770 in exon 7, causing a threonine-to-methionine substitution at amino acid 257 of the protein (T257M). This mutation has been observed in homozygosity in a patient nonresponsive to the cofactor treatment. The third mutation, found in heterozygosity in a patient responsive to pyridoxine treatment, is an insertion of 68 bp in exon 8 at base 844, which introduces a premature termination codon. The fourth mutation is C-to-T transition in exon 2 at base 262, causing a proline-to-serine substitution at position 88 of the protein (P88S). This mutation is carried on a single allele in three affected sisters responsive to the cofactor treatment. In addition, six previously reported mutations (A114V, E131D, P145L, I278T, G307S, and A1224-2C) have been tested in 14 independent Italian families. Mutations A114V and I278T are carried by three and by seven independent alleles, respectively. The other four mutations--including G307S and A1224-2C, common among northern European patients--have not been detected.
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PMID:The molecular basis of homocystinuria due to cystathionine beta-synthase deficiency in Italian families, and report of four novel mutations. 776 55

Cystathionine beta-synthase (CBS) purification from mammalian tissues is complicated by proteolysis and enzyme aggregation. To surmount these difficulties, we cloned human CBS cDNA in tandem with the beta-galactosidase sequence of the fusion vector, pAX5-, then expressed the fusion protein, beta-galactosidase/CBS, in transformed Escherichia coli cells. Proteolytic treatment of the ammonium sulfate fraction of bacterial lysates with endoproteinase Xa liberated CBS which could then be separated from its fusion partner by DEAE-cellulose chromatography. This nearly homogeneous enzyme preparation was purified 140-fold over the crude bacterial lysate with nearly 50% recovery, and its specific activity, 210 U/mg protein, was comparable to that purified from human liver. The purified enzyme contained pyridoxal 5'-phosphate and exhibited positive cooperativity toward S-adenosyl-L-methionine (Hill coefficient = 5.2; Kact = 34 microM). Km values of the cloned enzyme in the absence of AdoMet are 3.1 and 1.1 mM for serine and homocysteine, respectively. They are virtually identical to those from human hepatic CBS. A Soret absorbance band (lambda max = 428 nm) which shifted to 448 nm after reduction with sodium dithionite revealed the presence of heme in the enzyme. Expression of the fusion protein in E. coli with subsequent purification represents the first time this enzyme has been isolated in sufficient quantities for biophysical and biochemical investigation.
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PMID:Expression of human cystathionine beta-synthase in Escherichia coli: purification and characterization. 782 2

Studies by various investigators have indicated that elevated levels of plasma homocyst(e)ine are strongly associated with the occurrence of occlusive vascular diseases. With the eventual aim of determining whether or not elevated plasma homocyst(e)ine concentrations are directly causative of cardiovascular diseases, we have generated mice that are moderately and severely homocyst(e)inemic. Homologous recombination in mouse embryonic stem cells was used to inactivate the cystathionine beta-synthase [L-serine hydrolyase (adding homocysteine), EC 4.2.1.22] gene. Homozygous mutants completely lacking cystathionine beta-synthase were born at the expected frequency from matings of heterozygotes, but they suffered from severe growth retardation and a majority of them died within 5 weeks after birth. Histological examination showed that the hepatocytes of homozygotes were enlarged, multinucleated, and filled with microvesicular lipid droplets. Plasma homocyst(e)ine levels of the homozygotes were approximately 40 times normal. These mice, therefore, represent a model for severe homocyst(e)inemia resulting from the complete lack of cystathionine beta-synthase. Heterozygous mutants have approximately 50% reduction in cystathionine beta-synthase mRNA and enzyme activity in the liver and have twice normal plasma homocyst(e)ine levels. Thus, the heterozygous mutants are promising for studying the in vivo role of elevated levels of homocyst(e)ine in the etiology of cardiovascular diseases.
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PMID:Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia. 787 23

Cystathionine beta-synthase (CBS) deficiency is the most common cause of homocystinuria in humans. The human gene maps to chromosome 21q22.3 and encodes the CBS subunit of 551 amino acid residues (63kDa). CBS, a tetramer of these subunits, binds its two substrates, homocysteine and serine, and three additional ligands: pyridoxal 5'-phosphate, S-adenosylmethionine, and haem. Screening for mutations by expressing patient cDNA segments in E. coli permitted us to separate the parental CBS alleles, localize each mutation within one third of the cDNA, and functionally analyse the mutant protein. Using this method we identified the first 14 mutations in homocystinuria. The most common mutation in patients of predominantly 'Celtic' origin is the G919A transition which substitutes serine for glycine 307.
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PMID:Komrower Lecture. Molecular basis of phenotype expression in homocystinuria. 796 89

Cystathionine beta-synthase (beta-CTSase), which catalyses cystathionine synthesis from serine and homocysteine, was purified to homogeneity from Saccharomyces cerevisiae. The molecular mass of the enzyme was estimated to be 235 kDa by gel filtration and 55 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that it is a homotetramer. The N-terminal amino acid sequence of the enzyme perfectly coincided with that deduced from the nucleotide sequence of CYS4, except for the absence of initiation The purified beta-CTSase catalysed cysteine synthesis from serine (or O-acetylserine) and H2S. From this finding, we discuss the multifunctional nature and evolutionary divergence of S-metabolizing enzymes.
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PMID:Purification and properties of Saccharomyces cerevisiae cystathionine beta-synthase. 801 3

The responsibility of cysteine synthase (EC 4.2.99.8) from watermelon (Citrullus vulgaris) for the formation of beta-(pyrazole-1-yl)-L-alanine, a non-protein amino acid specifically accumulated in Curcubitaceae plants, was confirmed in vitro and in vivo by the cloned cDNA on expression vectors, pCCS11 and pCEN1. The cDNA sequence derived from pCCS11, an expression vector driven by the lacZ promoter, was placed under the transcriptional control of strong T7 promoter of pET3d to yield an over-expression vector, pCEN1, in Escherichia coli. The concentration of the exogenous cysteine synthase protein was increased up to approximately 10% of the total soluble protein of E. coli cells by the expression of cDNA on pCEN1. beta-(Pyrazole-1-yl)-L-alanine was formed in vitro from O-acetyl-L-serine and pyrazole by the action of cysteine synthase expressed in E. coli carrying pCCS11 or pCEN1. To confirm the responsibility of cysteine synthase for the formation of beta-(pyrazole-1-yl)-L-alanine in vivo, the feeding experiments of pyrazole and serine or O-acetyl-L-serine were carried out using the transformed E. coli culture. beta-(Pyrazole-1-yl)-L-alanine was produced in vivo by feeding the substrates to the culture of E. coli carrying pCEN1. These results provide the confirming evidence that the cloned cysteine synthase of watermelon catalyzes the formation of beta-(pyrazole-1-yl)-L-alanine, indicating that beta-pyrazolealanine synthase is identical with cysteine synthase in Cucurbitaceae plants.
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PMID:Evidence for identity of beta-pyrazolealanine synthase with cysteine synthase in watermelon: formation of beta-pyrazole-alanine by cloned cysteine synthase in vitro and in vivo. 828 Jan 25

Homocysteine can be methylated to form methionine by the cobalamin- (Cbl) and folate-dependent enzyme, methionine synthase; serum levels of total homocysteine are elevated in greater than 95% of patients with either Cbl or folate deficiency. Homocysteine can also condense with serine to form cystathionine in a pyridoxal phosphate-dependent reaction catalyzed by cystathionine beta-synthase. Cystathionine is subsequently cleaved to cysteine and alpha-ketobutyrate by the pyridoxal phosphate-dependent enzyme gamma-cystathionase. To assess levels of cystathionine in Cbl and folate deficiency, we developed a new capillary gas chromatographic-mass spectrometric assay and measured cystathionine in the serum of normal subjects and patients with clinically confirmed deficiencies of these vitamins. The normal range for serum cystathionine was 65 to 301 nmol/L (median = 126 nmol/L) for 50 normal blood donors. In 30 patients with clinically confirmed Cbl deficiency, values for cystathionine ranged from 208 nmol/L to 2,920 nmol/L (median = 816 nmol/L) and 26 (87%) had levels above the normal range. In 20 patients with clinically confirmed folate deficiency, cystathionine concentrations ranged from 138 nmol/L to 4,150 nmol/L (median = 1,560 nmol/L) and 19 (95%) had values above the normal range. Five homozygotes for cystathionine beta-synthase deficiency had high values for serum-total homocysteine and low or low-normal values for serum cystathionine that ranged from 30 nmol/L to 114 nmol/L even though they were on treatment with pyridoxine and had partially responded. One patient with a defect in the synthesis of 5-CH3-tetrahydrofolate and five patients with defects in the synthesis of CH3-Cbl had high values for serum-total homocysteine and high values for cystathionine that ranged from 311 nmol/L to 1,500 nmol/L even though they were on treatment with folic acid and Cbl, respectively, and had partially responded. We conclude that levels of cystathionine are evaluated in the serum of most patients with Cbl and folate deficiency and that they are useful in the differential diagnosis of an elevated serum-total homocysteine level.
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PMID:Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency. 850 76

The free-living nematode Panagrellus redivivus can be used as a biochemical model for parasitic nematodes in the search for new chemotherapeutic agents. A novel cystathionine beta-synthase has been purified 3600-fold from the cytosol of P. redivivus. The enzyme catalyses the synthesis of cystathionine from homocysteine plus serine or cysteine. The enzyme, native M(r) 71.7 kDa, pI 4.7, is a dimer and also catalyses the replacement of the beta-SH group of cysteine with 2-mercaptoethanol to yield a thioether, S-(2-hydroxyethyl) cysteine and H2S. This reaction proceeds much faster than cystathionine synthesis and L-cysteine cannot be replaced by D-cysteine, L-cystine, N-acetyl L-cysteine, cysteamine of D,L-homocysteine. 2-Mercaptoethanol in the assay can be replaced by monothiolglycerol and to a lesser extent by cysteamine. The absolute K(m) values for L-cysteine and 2-mercaptoethanol were 0.13 +/- 0.05 mM and 1.72 +/- 0.24 mM, respectively, the absolute V(max) was 55 +/- 4.9 mumol.min(-1).mg protein(-1). The enzyme had a pH optimum of approx. 8.5 and did not require metal ions for activity. The enzyme was inhibited by a series of substrate analogues, anthelmintics and plant phenols. The P. redivivus enzyme differs markedly from its mammalian equivalent and suggests distinctive differences in sulphur amino acid metabolism in nematodes.
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PMID:A novel cystathionine beta-synthase from Panagrellus redivivus (Nematoda). 869 99

The secondary structural composition and substrate-induced conformational changes of recombinant human cystathionine beta-synthase (CBS) in aqueous solution have been investigated in its full-length form (tetramer of 63-kDa subunits) by Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopies. In addition, structural comparison of a proteolytic truncated form (dimer of 45-kDa subunits) to that of the full-length enzyme has also been carried out. Second-derivative and Fourier self-deconvolutional enhanced infrared spectra revealed amide I band components ascribed to beta-sheet (1689, 1638, and 1627 cm(-1)), alpha-helix (1658 cm(-1)), beta-turn (1679 and 1668 cm(-1)), and unordered (1651 cm(-1)) structures in the spectra of the full-length enzyme. Quantitative analysis of FT-IR and CD spectra reveals that the full-length enzyme consists of about 48-53% beta-sheet, 25-30% alpha-helix, 8-10% turn, and 10-19% unordered structures. Under constraint of the spectroscopic data, theoretical prediction of locations of these secondary structural elements using Garnier's method shows that human CBS may contain a beta-sheet/alpha-helix/beta-sheet core structure. Second-derivative spectrum of the truncated enzyme exhibited all the major spectral features that are present in the full-length enzyme, indicating a preservation of the core structure of the enzyme. Significant differences were observed between the infrared spectra of the enzymes with or without the substrate, serine, indicating a substrate-induced conformational change in the enzyme, which did not result in a change in overall composition of secondary structural content based on quantitative analysis of FT-IR and far-UV CD spectra.
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PMID:Secondary structure of recombinant human cystathionine beta-synthase in aqueous solution: effect of ligand binding and proteolytic truncation. 924 89


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