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)

Cystathionine beta-synthase (CBS) is a unique heme enzyme that catalyzes a PLP-dependent condensation of serine and homocysteine to give cystathionine. Deficiency of CBS leads to homocystinuria, an autosomal recessively inherited disease of sulfur metabolism. A truncated form of CBS in which the C-terminal amino-acid residues have been deleted has been prepared. The truncated CBS subunits form a dimer, in contrast to the full-length subunits which form tetramers and higher oligomers. The truncated CBS yielded crystals diffracting to 2.6 A which belong to space group P3(1) or P3(2). This is the first comprehensive structural investigation of a PLP and heme-containing enzyme.
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PMID:Crystallization and preliminary X-ray diffraction analysis of the active core of human recombinant cystathionine beta-synthase: an enzyme involved in vascular disease. 1117 83

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium. L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited O-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T. thermophilus HB8.
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PMID:Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8. 1122 9

Cystathionine beta-synthase (CBS) catalyses the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. Human CBS encodes five distinct 5' non-coding exons, the most frequent termed CBS -1a and CBS -1b, each transcribed from its own unique GC-rich TATA-less promoter. The minimal transcriptional region (-3792 to -3667) of the CBS -1b promoter was defined by 5'- and 3'-deletions, and transient transfections of reporter gene constructs in HepG2 cells, characterized by CBS transcription exclusively from the -1b promoter. Included in this 125 bp region are 3 GC-boxes (termed GC-a, GC-b and GC-c), an inverted CAAT-box and an E-box. By gel-shift and supershift assays, binding of specificity protein (Sp)1 and Sp3 to the GC-box elements, upstream stimulatory factor 1 (USF-1) to the E-box, and both nuclear factor (NF)-Y and an NF-1-like factor to the CAAT box could be demonstrated. By transient trans fections and reporter gene assays in HepG2 and Drosophila SL2 cells, a functional interplay was indicated between NF-Y binding to the CAAT-box, or between USF-1 binding to the E-box, and Sp1/Sp3 binding to the GC-box elements. In SL2 cells, NF-Y and Sp1/Sp3 were synergistic. Furthermore, both Sp1 and the long Sp3 isoform transactivated the CBS -1b minimal promoter; however, the short Sp3 isoforms were potent repressors. These results may explain the cell- or tissue-specific regulation of CBS transcription, and clarify the bases for alterations in CBS gene expression in human disease and Down's syndrome.
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PMID:Transcriptional regulation of the human cystathionine beta-synthase -1b basal promoter: synergistic transactivation by transcription factors NF-Y and Sp1/Sp3. 1141 40

Cystathionine beta-synthase (CBS) is a unique heme- containing enzyme that catalyzes a pyridoxal 5'-phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine. Deficiency of CBS leads to homocystinuria, an inherited disease of sulfur metabolism characterized by increased levels of the toxic metabolite homocysteine. Here we present the X-ray crystal structure of a truncated form of the enzyme. CBS shares the same fold with O-acetylserine sulfhydrylase but it contains an additional N-terminal heme binding site. This heme binding motif together with a spatially adjacent oxidoreductase active site motif could explain the regulation of its enzyme activity by redox changes.
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PMID:Structure of human cystathionine beta-synthase: a unique pyridoxal 5'-phosphate-dependent heme protein. 1148 94

Cystathionine beta-synthase (CBS), condensing homocysteine and serine, represents a key regulatory point in the biosynthesis of cysteine via the transsulfuration pathway. Inherited deficiency of CBS causes homocystinuria. CBS is activated by S-adenosyl-L-methionine (AdoMet) by inducing a conformational change involving a noncatalytic C-terminal region spanning residues 414-551. We report the purification of two patient-derived C-terminal mutant forms of CBS, S466L and I435T, that provide new insight into the mechanism of CBS regulation and indicate a regulatory function for the "CBS domain". Both of these point mutations confer catalytically active proteins. The I435T protein is AdoMet inducible but is 10-fold less responsive than wild-type (WT) CBS to physiologically relevant concentrations of this compound. The S466L form does not respond to AdoMet but is constitutively activated to a level intermediate between those of WT CBS in the presence and absence of AdoMet. Both mutant proteins are able to bind AdoMet, indicating that their impairment is related to their ability to assume the fully activated conformation that AdoMet induces in WT CBS. We found that I435T and WT CBS can be activated by partial thermal denaturation but that the AdoMet-stimulated WT, S466L, and a truncated form of CBS lacking the C-terminal region cannot be further activated by this treatment. Tryptophan and PLP fluorescence data for these different forms of CBS indicate that activation by AdoMet, limited proteolysis, and thermal denaturation share a common mechanism involving the displacement of an autoinhibitory domain located in the C-terminal region of the protein.
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PMID:Regulation of human cystathionine beta-synthase by S-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region. 1152 6

Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.
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PMID:The reaction of yeast cystathionine beta-synthase is rate-limited by the conversion of aminoacrylate to cystathionine. 1153 64

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously characterized the CBS -1b minimal promoter (-3792 to -3667) and found that Sp1/Sp3, nuclear factor Y, and USF-1 were involved in the regulation of basal promoter activity (Ge, Y., Konrad, M. A., Matherly, L. H., Taub, J. W. (2001) Biochem. J. 357, 97-105). In this study, the critical cis-elements and transcription factors in the CBS -1b upstream region (-4046 to -3792) were examined in HT1080 and HepG2 cells, which differ approximately 10-fold in levels of CBS transcripts transcribed from the CBS -1b promoter. In DNase I footprint and gel shift analyses and transient transfections of mutant CBS -1b promoter constructs into HT1080 and HepG2 cells, transcriptionally important roles for Sp1/Sp3 binding to three GC boxes and one GT box and for binding of myeloid zinc finger 1-like proteins to two myeloid zinc finger 1 elements were indicated. In gel shift assays, very low levels of Sp1/Sp3 DNA-protein complexes were detected in HT1080 cells compared with HepG2 cells despite comparable levels of nuclear factor Y and USF-1 binding and similar levels of Sp1 and Sp3 proteins on Western blots. Mixing of HT1080 and HepG2 nuclear extracts resulted in no difference in total Sp factor binding in gel shift assays, thus excluding a role for an unknown activator or inhibitor in the disparate Sp1/Sp3 binding between the lines. Increased Sp1/Sp3 binding in gel shift assays was observed upon treatment of HT1080 nuclear extracts with protein kinase A, and decreased Sp1/Sp3 binding resulted from treatment of HepG2 nuclear extracts with calf alkaline phosphatase, suggesting a role for changes in Sp1/Sp3 phosphorylation in transcription factor binding and transactivation of the CBS -1b promoter. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down syndrome.
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PMID:Transcriptional regulation of cell-specific expression of the human cystathionine beta -synthase gene by differential binding of Sp1/Sp3 to the -1b promoter. 1156 58

Sulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified. Here we describe the regulation of the lactococcal metC-cysK operon, encoding a cystathionine beta-lyase (metC) and cysteine synthase (cysK). Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression. Other sulfur sources tested showed no significant effect on metC-cysK gene expression. In addition we found that O-acetyl-l-serine, the substrate of cysteine synthase, was an inducer of the metC-cysK operon. Using a random mutagenesis approach, we identified two genes, cmbR and cmbT, involved in regulation of metC-cysK expression. The cmbT gene is predicted to encode a transport protein, but its precise role in regulation remains unclear. Disruption of cmbT resulted in a two- to threefold reduction of metC-cysK transcription. A 5.7-kb region containing the cmbR gene was cloned and sequenced. The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of the metC-cysK operon. In analogy to CysB from Escherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of the metC-cysK operon.
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PMID:Regulation of the metC-cysK operon, involved in sulfur metabolism in Lactococcus lactis. 1174 47

Our studies of the reaction mechanism of cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme. The enzyme catalyzes the reaction of L-serine with L-homocysteine to form L-cystathionine through a series of pyridoxal phosphate intermediates. In this work, we explore the substrate specificity of the enzyme by use of substrate analogues combined with kinetic measurements under pre-steady-state conditions and with circular dichroism and fluorescence spectroscopy under steady-state conditions. Our results show that L-allothreonine, but not L-threonine, serves as an effective substrate. L-Allothreonine reacts with the pyridoxal phosphate cofactor to form a stable 3-methyl aminoacrylate intermediate that absorbs maximally at 446 nm. The rapid-scanning stopped-flow results show that the binding of L-allothreonine as the external aldimine is faster than formation of the 3-methyl aminoacrylate intermediate. The 3-methyl aminoacrylate intermediate reacts with L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine, which was characterized by nuclear magnetic resonance spectroscopy. This new amino acid may be a useful analogue of L-cystathionine.
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PMID:Yeast cystathionine beta-synthase reacts with L-allothreonine, a non-natural substrate, and L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine. 1182 27

Human cystathionine beta-synthase is a hemeprotein that catalyzes a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine into cystathionine. Biophysical characterization of this enzyme has led to the assignment of the heme ligands as histidine and cysteinate, respectively, which has recently been confirmed by crystal structure determination of the catalytic core of the protein. Using site-directed mutagenesis, we confirm that C52 and H65 represent the thiolate and histidine ligands to the heme. Conversion of C52 to alanine or serine results in spectral properties of the resulting hemeprotein that are consistent with the loss of a thiolate ligand. Thus, the Soret peak blue-shifts from 428 to 415 and 417 nm in the ferric forms of the C52S and C52A mutants, respectively, and from 450 to 423 nm in the ferrous states of both mutants. Addition of CO to the dithionite-reduced ferrous C52 mutants results in spectra with Soret peaks at 420 nm. EPR spectroscopy of the ferric C52 variants reveals the predominance of a high-spin species. The H65R mutant, a variant described in a homocystinuric patient, has Soret peaks at 424, 421, and 420 nm in the ferric, ferrous, and ferrous CO states, respectively. EPR spectroscopy reveals predominance of the low-spin species. Both C52A and C52S mutations lead to protein with substoichiometric heme (19% with respect to wild type); however, the PLP content is comparable to that of wild-type enzyme. The heme and PLP contents of the H65R mutant are 40% and 75% that of wild-type enzyme. These results indicate that heme saturation does not dictate PLP saturation in these mutant enzymes. Both H65 and C52 variants display low catalytic activity, revealing that changes in the heme binding domain modulate activity, consistent with a regulatory role for this cofactor.
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PMID:Effects of heme ligand mutations including a pathogenic variant, H65R, on the properties of human cystathionine beta-synthase. 1192 27


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