Expression of Human Cystathionine β-Synthase in Escherichia coli: Purification and Characterization

Bukovská, Gabriela; Kéry, Vladimír; Kraus, Jan P.

doi:10.1006/prep.1994.1063

Cited by 80 publications

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“…The cDNA portion of the previously constructed pAX5 − HCBS WT vector [16] encoding CBS amino acids 1-551 was cut out using ApaI and SalI restriction endonucleases. Finally, the pGEX-6P-1 vector cassette, the two hybridized primers and the fragment cut out of the pAX5 − HCBS WT vector were ligated.…”

Section: Nih Public Accessmentioning

confidence: 99%

“…4M sodium chloride was added immediately to a final concentration of 10 mM, and the mixture was allowed to slowly cool at room temperature. Under these conditions, the two primers hybridize while forming a short double-stranded insert with ApaI-ApaI overhangs.The cDNA portion of the previously constructed pAX5 − HCBS WT vector [16] encoding CBS amino acids 1-551 was cut out using ApaI and SalI restriction endonucleases. Finally, the pGEX-6P-1 vector cassette, the two hybridized primers and the fragment cut out of the pAX5 − HCBS WT vector were ligated.…”

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confidence: 99%

“…Previously, an Escherichia coli expression system using β-galactosidase (β-gal) was used for the purification of CBS [16]. This system had several disadvantages.…”

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confidence: 99%

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Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

Frank

Kent

Meier

et al. 2008

Archives of Biochemistry and Biophysics

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In this paper, we describe the expression and characterization of recombinant human cystathionine β-synthase (CBS) in Escherichia coli. We have used a glutathione-S-transferase (GST) fusion protein vector and incorporated a cleavage site with a long hinge region which allows for the independent folding of CBS and its fusion partner. In addition, our construct has the added benefit of yielding a purified CBS which only contains one extra glycine amino acid residue at the Nterminus. In our two-step purification procedure we are able to obtain a highly pure enzyme in sufficient quantities for crystallography and other physical chemical methods. We have investigated the biochemical and catalytic properties of purified full-length human CBS and of two truncation mutants lacking the Cterminal domain or both the N-terminal heme-binding and the C-terminal regulatory regions. Specifically, we have determined the pH optima of the different CBS forms and their kinetic and spectral properties. The full-length and the C-terminally truncated enzyme had a broad pH 8.5 optimum while the pH optimum of the N-and C-terminally truncated enzyme was sharp and shifted to pH 9. Furthermore, we have shown unequivocally that CBS binds one mole of heme per subunit by determining both the heme and the iron content of the enzyme. The activity of the enzyme was unaffected by the redox status of the heme iron. Finally, we show that CBS is stimulated by S-adenosyl-L-methionine but not its analogs. KeywordsHomocystinuria; S-adenosylmethionine; Pyridoxal-5′ phosphate; Heme Cystathionine β-synthase (CBS) 2 (L-Serine hydrolyase (adding homocysteine), EC 4.2.1.22) is central to sulfur amino acid metabolism in eukaryotes, and complete CBS deficiency is the principal cause of homocystinuria in humans. The CBS-catalyzed condensation of serine and homocysteine generates cystathionine which is then cleaved with cystathionine γ-lyase (CGL) to yield cysteine [1].Cysteine synthase (O-acetylserine sulfhydrase, OASS; CS), the CBS counterpart in prokaryotes, plants and enteric protozoa, catalyzes the formation of cysteine from Oacetylserine and hydrogen sulfide (H 2 S) with the concomitant release of acetic acid. Unlike mammals, these organisms are able to assimilate inorganic sulfur into organic sulfur and are therefore essential players in the sulfur cycle in nature. CS and CBS are affiliated with the large β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes and have a high degree * Corresponding author. Fax: +1 303 724 3838. Jan.Kraus@UCHSC.edu (J.P. Kraus). 1 These authors contributed equally to this work.2 Abbreviations used: CBS, Cystathionine β-synthase; GST, glutathione S-transferase; PLP, pyridoxal 5′-phosphate; AdoMet, Sadenosyl-L-methionine; CS, cysteine synthase; OASS, O-acetylserine sulfhydrase; β-gal, β-galactosidase; OAS, O-acetyl serine; AdoHcy, S-adenosylhomocysteine. CBS is the only PLP dependent enzyme which contains heme [7]. The heme is co-ordinated by Cys 52 and His 65 residues located in the N-terminal region of the enzym...

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Section: Nih Public Accessmentioning

confidence: 99%

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confidence: 99%

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Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

Frank

Kent

Meier

et al. 2008

Archives of Biochemistry and Biophysics

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“…[13][14][15] Among these VSCs, endogenous hydrogen sulfide (H2S) is synthesized from most tissues in mammals, 16,17 for which two enzymes are involved in this synthesis, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). [18][19][20][21] To prove the hypothesis that esophago-gastric mucosa might be the principal source for VSCs and diseased mucosa might be associated with halitosis, we measured the VSCs in either gastric juices or breath air and correlated the association between erosive mucosa and the levels of VSC.…”

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confidence: 99%

Volatile Sulfur Compounds as a Predictor for Esophagogastroduodenal Mucosal Injury

Yoo

Jung²,

Sohn³

et al. 2008

Gut Liver

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Background/Aims: Halitosis is a symptom that bothers patients more socially than medically and its pathogenic mechanisms are unclear and treatment armamenterium is limited. Clinicians generally ignored active interventions. Since halitosis is closely associated with volatile sulfur compounds (VSCs), we used a Halimeter and gas chromatography to measure VSCs in patients with Helicobacter-pylori (H. pylori)-associated gastric diseases. Methods: We categorized 72 patients with H. pylori infection into two groups based on their endoscopic findings: a nonerosive mucosal group (NE, n=24) and an erosive mucosal group (E, n=48). Halitosis was objectively assessed by applying either a Halimeter to breath air or gas chromatography to gastric juice. Simultaneously, the expression of VSC-generating enzyme was measured with reverse-transcriptase PCR using mRNA isolated from biopsy tissues. Results: The levels of VSCs in exhaled breaths or aspirated gastric juices differed significantly between the NE and E groups (p ＜0.00001), suggesting that VSCs might reflect eroded epithelial damage induced by H. pylori infection. The expressions of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) were broadly consistent with the degree of mucosal injury. Conclusions: Erosive changes in esophagogastroduodenal mucosa were strongly correlated with increased VSC levels, suggesting that halitosis might result from H. pylori-associated erosive lesions. (Gut and Liver 2008;2: 113-118)

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“…The human CBS gene product is a 63,000-Da polypeptide. The tetrameric protein is catalytically dependent on both heme and pyridoxal phosphate and is allosterically regulated by S-adenosylmethionine (1)(2)(3)(4). The human CBS gene spans over 30 kilobases and consists of 23 exons ranging in size from 42 to 209 base pairs (5).…”

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Transcriptional Regulation of Cell-specific Expression of the Human Cystathionine β-Synthase Gene by Differential Binding of Sp1/Sp3 to the −1b Promoter

Matherly¹,

Taub²

2001

Journal of Biological Chemistry

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Cystathionine ␤-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, Cystathionine ␤-synthase (CBS 1 ; EC 4.2.1.22), a pyridoxal 5Ј-phosphate-dependent enzyme involved in the transsulfuration pathway, catalyzes the condensation of L-serine and Lhomocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. The human CBS gene product is a 63,000-Da polypeptide. The tetrameric protein is catalytically dependent on both heme and pyridoxal phosphate and is allosterically regulated by S-adenosylmethionine (1-4). The human CBS gene spans over 30 kilobases and consists of 23 exons ranging in size from 42 to 209 base pairs (5). The CBS polypeptide is encoded by exons 1-14 and 16. The human CBS gene encodes multiple mRNAs differing in their 5Ј-untranslated regions, resulting from the use of five alternative noncoding exons (designated Ϫ1a to Ϫ1e) and a constant exon 0. Transcripts containing exons Ϫ1a and Ϫ1b appear to be the most abundant and are found in an assortment of adult and fetal tissues (6). By contrast, use of exons Ϫ1c, Ϫ1d, and Ϫ1e appears to be rare. There are at least two GC-rich TATA-less promoters upstream of exons Ϫ1a and Ϫ1b, containing numerous putative transcription elements (Sp1 (specificity protein 1), AP1, AP2, etc.) (5, 6). In our recent study of the CBS Ϫ1b minimal promoter (mapping between positions Ϫ3792 and Ϫ3667), we demonstrated important transactivating roles for Sp1, Sp3, NF-Y, and USF-1 (7).YThe CBS gene has been localized to human chromosome 21 (e.g. 21q22.3), and its overexpression has been suggested to be linked to certain of the phenotypic features of Down syndrome (DS). Elevated CBS expression in DS results in low plasma homocysteine compared with non-DS individuals and has been suggested to contribute to decreased atherosclerosis in DS patients (8). In our own studies of CBS and DS, we also found striking increases (ϳ12-fold) in CBS transcripts in myeloblasts from DS children with AML compared with non-DS myeloblasts (9). Interestingly, this elevated CBS expression was associated with increased in vitro sensitivities to cytosine arabinoside (Ara-C) and generation of Ara-C triphosphate, likely due to downstream effects of CBS on endogenous folate and nucleotide pools (9 -12). This may explain, in part, the remarkably high event-free survival rates (70 -100%) and low relapse rates (Ͻ15%) of DS children with AML compared with non-DS children treated with Ara-C-based chemotherapy protocols. Increased CBS transcripts in DS over non-DS myeloblasts in-*

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Expression of Human Cystathionine β-Synthase in Escherichia coli: Purification and Characterization

Cited by 80 publications

References 0 publications

Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

Volatile Sulfur Compounds as a Predictor for Esophagogastroduodenal Mucosal Injury

Transcriptional Regulation of Cell-specific Expression of the Human Cystathionine β-Synthase Gene by Differential Binding of Sp1/Sp3 to the −1b Promoter

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