• Title/Summary/Keyword: cystathionine

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Regulation of Enzymes Involved in Methionine Biosynthesis in Corynebacterium glutamicum

  • Yeom, Hye-Jin;Hwang, Byung-Joon;Lee, Myong-Sok;Kim, Youn-Hee;Lee, Heung-Shick
    • Journal of Microbiology and Biotechnology
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    • v.14 no.2
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    • pp.373-378
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    • 2004
  • The regulatory mechanism of methionine biosynthesis in Corynebacterium glutamicum was analyzed at the protein arid gene expression level. O-Acetylhomoserine sulfhydraylase (encoded by metY) was inhibited by 10 mM methionine to a residual activity of 10% level, whereas no such inhibition was found with cystathionine $\gamma$-synthase (encoded by metB) and cystathionine $\beta$-lyase (encoded by metC). The enzymatic activity of homoserine acetyltransferase (encoded by metX) was repressed to a residual activity of 25% level by 10 mM methionine which was added to the growth medium. Cystathionine $\gamma$-synthase and cystathionine $\beta$-lyase were also repressed by 10 mM methionine, but only to a residual activity of 50-70% level. O-Acetylhomoserine sulfhydrylase was very sensitive to repression by 10 mM methionine, showing residual activity of 13%. In addition, homoserine acetyltransferase was also repressed by 10 mM cysteine to 50% of its original activity. No repression of the enzymes by S-adenosyl methionine was observed. The pattern of repression by methionine indicated that the metB and aecD genes might be regulated by a common mechanism, while the metA and metY genes are differently regulated.

Direct Conversion of L-Selenomethionine into Methylselenol by Human Cystathionine ${\gamma}$-Lyase (인간 Cystathionine ${\gamma}$-Lyase에 의한 Selenomethionine의 Methylselenol로의 직접분해)

  • Cho, Hyun-Nam;Jhee, Kwang-Hwan
    • Microbiology and Biotechnology Letters
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    • v.42 no.1
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    • pp.11-17
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    • 2014
  • Selenium is an essential trace element for mammals, but it is very toxic. Therefore, the control of selenium concentrations should be precisely and effectively monitored. Selenium is naturally obtained through foods and seleno-L-methionine (LSeMet) is a major form of selenium. It has been reported that L-SeMet is only converted into Se-adenosyl-L-SeMet. However, a recent study suggested that L-SeMet was directly metabolized into methylselenol ($CH_3SeH$) in mouse liver extract by the reaction of cystathionine ${\gamma}$-lyase (CGL). The canonical reaction of CGL was known to catalyze the cleavage of L-cystathionine to L-cysteine, ${\alpha}$-ketobutyrate and $NH_3$. In the present study, we found that L-SeMet could be directly converted to $CH_3SeH$ using purified homogenous human CGL instead of mouse liver cytosol. Authentic $CH_3SeH$ was prepared by reduction of dimethyldiselenide with sodium tetrahydroborate. The gaseous product of the enzymatic reaction with L-SeMet was analyzed by GC/MS spectrometry. The GC/MS data was identical to that of authentic dinitrophenyl selenoether. We also analyzed the kinetic parameters for the formation of $CH_3SeH$ from L-SeMet by human and mouse CGL. These results suggest that human CGL is a critical enzyme which is responsible for L-SeMet metabolism.

Myrrha-induced Apoptosis in Human Cervical Carcinoma HeLa Cells (몰약(沒藥)이 자궁경부암세포(子宮經部癌細胞)(HeLa Cell)의 Apoptosis에 미치는 영향(影響))

  • Park, Jong-Kyu;Jo, Ok-Hyon;Kim, Song-Baeg;Cho, Han-Baek
    • The Journal of Korean Obstetrics and Gynecology
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    • v.19 no.1
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    • pp.97-110
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    • 2006
  • Purpose : To address the ability of Myrrha (MY) to induce cell death, we investigated the effect of MY on apoptosis. In human cervical carcinoma HeLa cells, apoptosis occurred following MY exposure in a dose-dependent manner. Methods : We have tested several kinds of anti-oxidants to investigate the MY-induced apoptotic mechanism. Among the anti-oxidants, N-acetyl cysteine(NAC) or reduced glutathione (GSH) protects MY-induced apoptosis. NAC is an aminothiol and synthetic precursor of intracellular cysteine and GSH. To confirm the role of GSH in MY-induced apoptosis, methionine and cystathionine-glutathione extrusion inhibitors were treated in the presence of MY. Results : NAC, GSH, methionine or cystathionine led to protective effect against MY-induced apoptosis in HeLa cells. The GSH and GSH-associated reagents regulate MY-induced cytochrome c release and the resultant caspase-3 activation. Furthermore, the two specific inhibitors of carrier-mediated GSH extrusion, methionine and cystathionine demonstrate GSH extrusion occurs via a specific mechanism. While decreasing GSH extrusion and protecting against MY-induced apoptosis, methionine and cystathionine failed to exert anti-apoptotic activity in cells previously deprived of GSH. Conclusion : the target of the protection is indeed GSH extrusion. This shows that the protective effect is achieved by forcing GSH to stay within the cells during apoptogenic treatment. All this evidence indicates the extrusion of GSH precedes andis responsible for the apoptosis, probably by altering the intracellular redox state, thus giving a rationale for the development of redox-dependent apoptosis in MY-treated human cervical carcinoma HeLa cells.

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Biochemical Characteristics for the Cofactor Free Mutant of Yeast Homocysteine Catalyzing Enzyme, Cystathionine ${\beta}$-Synthase (조효소를 함유하지 않는 효모의 Homocysteine 분해효소, Cystathionine ${\beta}$-Synthase의 생화학적 특성)

  • Jhee, Kwang-Hwan;Cho, Hyun-Nam;Yang, Seun-Ah;Lee, In-Seun
    • Microbiology and Biotechnology Letters
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    • v.35 no.3
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    • pp.196-202
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    • 2007
  • Mutations in the cystathionine ${\beta}$-synthase (CBS) gene cause homocystinuria, the most frequent inherited disorder in sulfur metabolism. CBS is the unique enzyme using both heme and pyridoxal 5-phosphate (PLP) for activity. Among the reported 140 mutations, one of the most common disease-causing alterations in human CBS is G307S mutation. To investigate the pathogenic mechanism of G307S by spectroscopic methods, we engineered the full length and the truncated G247S mutation of yeast CBS that is corresponding mutation to human G307S. Yeast CBS does not contain heme and thus gives a merit to study the spectroscopic properties. The UV-visible spectra of the purified full length and the truncated G247S yeast CBSs showed the total absence of PLP in the protein. The absence of PLP in G247S mutation was also confirmed by the PLP-cyanide adduct formation experiment, which was conducted by the incubation of the purified enzyme with KCN. The adducts were detected using a circular dichroism (CD) and a spectrofluorimeter. Radio isotope activity assay of full length and truncated G247S proteins also gave no activity. Our yeast G247S mutation data suggested that G307S might make the distortion of the active site so that cofactor PLP and substrate can not fit inside the active site. Our yeast CBS study addressed the reason why the G307S mutation in human CBS makes the enzyme inactive that consequently leads to severe clinical phenotype.

Overexpression and Activity Analysis of Cystathionine γ-Lyase Responsible for the Biogenesis of H2S Neurotransmitter (새로운 신경전달물질 H2S 발생 효소, cystathionine γ-lyase의 대량발현 조건과 활성측정)

  • Kim, Kyoung-Ran;Byun, Hae-Jung;Cho, Hyun-Nam;Kim, Jung-Hyun;Yang, Seun-Ah;Jhee, Kwang-Hwan
    • Journal of Life Science
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    • v.21 no.1
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    • pp.119-126
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    • 2011
  • There is a growing recognition of the significance of $H_2S$ as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine ${\beta}$-synthase (CBS) and cystathionine ${\gamma}$-lyase (CGL), are believed to be chiefly responsible for $H_2S$ biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down's syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for ${\alpha}-{\gamma}$ elimination reaction with L-cystathionine and 88% for ${\alpha}-{\beta}$ elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 nm absorption maximum, which is attributed to the binding cofactor, pyridoxal 5'-phosphate (PLP).

Disruption of the metC Gene Affects Methionine Biosynthesis in Pectobacterium carotovorum subsp. carotovorum Pcc21 and Reduces Soft-Rot Disease

  • Seonmi, Yu;Jihee, Kang;Eui-Hwan, Chung;Yunho, Lee
    • The Plant Pathology Journal
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    • v.39 no.1
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    • pp.62-74
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    • 2023
  • Plant pathogenic Pectobacterium species cause severe soft rot/blackleg diseases in many economically important crops worldwide. Pectobacterium utilizes plant cell wall degrading enzymes (PCWDEs) as the main virulence determinants for its pathogenicity. In this study, we screened a random mutant, M29 is a transposon insertion mutation in the metC gene encoding cystathionine β-lyase that catalyzes cystathionine to homocysteine at the penultimate step in methionine biosynthesis. M29 became a methionine auxotroph and resulted in growth defects in methionine-limited conditions. Impaired growth was restored with exogenous methionine or homocysteine rather than cystathionine. The mutant exhibited reduced soft rot symptoms in Chinese cabbages and potato tubers, maintaining activities of PCWDEs and swimming motility. The mutant was unable to proliferate in both Chinese cabbages and potato tubers. The reduced virulence was partially restored by a complemented strain or 100 µM of methionine, whereas it was fully restored by the extremely high concentration (1 mM). Our transcriptomic analysis showed that genes involved in methionine biosynthesis or transporter were downregulated in the mutant. Our results demonstrate that MetC is important for methionine biosynthesis and transporter and influences its virulence through Pcc21 multiplication in plant hosts.

Regulation of methionine biosynthesis in plants; transgenic study

  • Kim, Jungsup;Thomas Leustek
    • Proceedings of the Botanical Society of Korea Conference
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    • 2002.04a
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    • pp.73-82
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    • 2002
  • The committing step in Met and S-adenosyl-L-methionine (SAM) synthesis is catalyzed by cystathionine ${\gamma}$ -synthase (CGS). Transgenic Arabidopsis thaliana overexpressing CGS under control of 35S promoter show increased soluble Met and its metabolite S-methylmethionine, but only at specific stages of development. CGS-overexpressing seedlings are resistant to ethionine. Similar results were obtained with transgenic potato plants overexpressing Arabidopsis CGS. Several of the transgenic lines show silencing of CGS resulting in deformed p]ants with a reduced capacity for reproductive growth similar as transgenic plants by antisense RNA (CGS[-]). Exogenous feeding of Met to the CGS[-] and CGS[+] silenced plants partially restores their growth. Similar morphological deformities are observed in plants cosuppressed for SAM synthetase, even though such plants accumulate 250 fold more soluble Met than wild type and they overexpress CGS. The results suggest that the abnormalities associated with CGS and SAM synthetase silencing are due in part to a reduced ability to produce SAM, and that SAM may be a regulator of CGS expression.

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Hepatic Metabolism of Sulfur Amino Acids During Septic Shock (패혈성 쇼크에서 간의 유황함유 아미노산 대사)

  • Kang, Keon-Wook;Kim, Sang-Kyum
    • YAKHAK HOEJI
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    • v.51 no.6
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    • pp.383-388
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    • 2007
  • It has been reported that sulfur-containing intermediates or products in the transsulfuration pathway including S-adenosylmethionine, 5'-methylthioadenosine, glutathione and taurine can prevent liver injury mediated by inflammation response induced by lipopolysaccharide (LPS) treatment. The present study examines the modulation of hepatic metabolism of sulfur amino acid in a model of acute sepsis induced by LPS treatment (5 mg/kg, iv). Serum TNF-alpha and hepatotoxic parameters were significantly increased in rats treated with LPS, indicating that LPS results in sepsis at the doses used in this study. LPS also induced oxidative stress determined by increases in malondialdehyde levels and decreases in total oxy-radical scavenging capacities. Hepatic methionine and glutathione concentrations were decreased, but S-adenosylho-mocysteine, cystathionine, cysteine, hypotaurine and taurine concentrations were increased. Hepatic protein expression of methionine adenosyltransferase, cystathionine beta-synthase and cysteine dioxygenase were induced, but gamma-glutamylcysteine ligase catalytic subunit levels were decreased. The results show that sepsis activates transsulfuration pathway from methionine to cysteine, suggesting an increased requirement for methionine during sepsis.