Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.2.1.22 (cystathionine beta-synthase)
 965 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The coding sequences of the cysE and cysK genes from Escherichia coli, which encode the enzymes of the cysteine biosynthetic pathway, namely, serine acetyltransferase (EC 2.3.1.30) and O-acetylserine sulfhydrylase (or cysteine synthase [EC 4.2.99.8]), were modified for expression in eukaryotic cells and introduced into murine L cells. A number of fusion genes comprising the cysE or cysK coding sequences joined to the promoter of the ovine metallothionein-Ia (MT-Ia) gene and various portions of the ovine growth hormone (GH) gene were prepared. Significant differences in the level of transcription were observed, depending on the amount and arrangement of the GH gene sequences used, the highest levels being obtained with the constructs MTCE10 and MTCK7, which contained only the GH 3' untranslated gene sequences. These two constructs were fused to produce the gene MTCEK1. In this single DNA sequence, each bacterial gene is under independent MT-Ia promoter control. Expression of the cysK sequence in this construct (MT-Ia promoter-cysE-3' GH sequence-MT-Ia promoter-cysK-3' GH sequence) was elevated compared with expression of the cysK gene in MTCK7. However, expression of the cysE sequence in MTCEK1 was only 40% of that of the cysE gene cloned into MTCE10. The double-promoter configuration, which enhances the expression of the second gene in MTCEK1, is proposed as a model for the modification of bacterial genes in general.
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PMID:Introduction and expression of the bacterial genes cysE and cysK in eukaryotic cells. 768 85

Subcellular localization and regulation of the spinach (Spinacia oleracea) cysteine synthase (O-acetyl-L-serine[thiol]-lyase, EC 4.2.99.8) isoforms (CysA, CysB, and CysC) were determined in transgenic tobacco (Nicotiana tabacum) and in spinach cell cultures. The 5' regions of CysB and CysC encoding the chloroplastic (CysB-TP) and the putative mitochondrial (CysC-TP) transit peptide (TP) sequences were fused to a bacterial beta-glucuronidase gene (gus) and expressed in tobacco under the control of the cauliflower mosaic virus 35S promoter. Subcellular fractionation of transgenic tobacco showed transportation of beta-glucuronidase proteins to chloroplasts by CysB-TP and to mitochondria by CysC-TP, respectively, indicating that both presequences were sufficient to act specifically as chloroplastic and mitochondrial TPs in vivo. The mRNA expression patterns of CysA (cytoplasmic form), CysB, and CysC genes under nitrogen- and sulfur-starved conditions were characterized in spinach cell cultures. In sulfur-starved cells, only slight differences (approximately 1.2- to 1.5-fold) in the mRNA levels of CysA and CysB were observed during the short-term (0-24 h) cultivation periods compared with cells grown in Murashige-Skoog medium. However, under nitrogen and nitrogen/sulfur double-deficient stress conditions, mRNA levels of CysC increased up to 500% of the original level within 72 h.
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PMID:Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur. 881 26

A sandwich ELISA method using peptide tags showing a specific affinity to a hydrophilic polystyrene surface (PS-tags), PS 19 composed of RAFIASRRIKRP and KPS19R10 of KRAFIASRRIRRP and a hydrophilic polystyrene (phi-PS) plate was used to analyze protein-protein interactions. An Escherichia coli cysteine synthase complex, in which serine acetyltransferase (SAT) interacts with O-acetylserine sulfhydrylase-A (OASS) was used as a model system. When the interaction was detected by the conventional sandwich ELISA method using a hydrophobic polystyrene (pho-PS) plate, for the exclusive use of ELISA, the signal intensity was barely detectable due to conformational change of the ligand protein, OASS in the adsorbed state. On the contrary, when OASS, genetically fused with PS19 (OASS-PS19) or chemically conjugated with KPS19R10 (OASS-KPS19R10), was immobilized on the phi-PS plate, a high signal intensity was detected. Furthermore, by applying the two-step sandwich ELISA, in which OASS-PS19 or OASS-KPS19R10 formed a complex with SAT in the blocking solution before immobilization on the phi-PS plate, the signal intensity was further increased with a much shorter operational time, because SAT in the blocking solution formed a complex with OASS-PS19 or OASS-KPS19R10 without any steric hindrance.
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PMID:Protein-protein interaction analysis using an affinity peptide tag and hydrophilic polystyrene plate. 1705 1

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.
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PMID:Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain. 1802

The aim of this study was to evaluate the possibility that synthetic forms of methionine-free alpha-casein and methionine-limited alpha casein could be produced by recombinant means to form the basis for developing an industrial-scale process for the provision of a foodstuff suitable for patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. As a first step, two forms of alpha casein gene, encoding methionine-free alpha casein (Fcas) or a methionine-limited alpha casein (Mcas), were synthesised and expressed in Escherichia coli. Using the overexpression vector pET28a, both genes were highly expressed in E. coli in soluble form as well as in inclusion bodies. The two recombinant proteins were purified by the one step methods using the fused His-tag and the Ni(2+)column and validated by Western blot analysis. This work paves the way for industrial-scale production of proteins suitable for patients with homocystinuria due to CBS deficiency.
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PMID:Production of synthetic methionine-free and synthetic methionine-limited alpha casein: protein foodstuff for patients with homocystinuria due to cystathionine beta-synthase deficiency. 2003 68