• 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.

• 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.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.619-619
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• 2005

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.581-581
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• 2003

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.384-384
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• 2005

• Journal of The Korean Society of Inherited Metabolic disease
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• v.15 no.2
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• pp.78-86
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• 2015

Purpose: Homocystinuria (OMIM#236200) is a metabolic disease caused by mutation in the CBS gene. This study was conducted to identify the clinical features and prognosis of homocystinuria as well as to find out the CBS gene mutations of the six homocystinuria patients who were receiving treatment in the Pediatric Department at Soonchunhyang University Hospital. Methods: From January 1992 to March 2015, clinical, biochemical, and genetic analyses were performed retrospectively on the six patients diagnosed with classic homocystinuria at Soonchunhyang University Hospital. Results: A total of six patients were included in this study, including three who were diagnosed with homocystinuria at the mean age of $50{\pm}22.5$ days based on their abnormal newborn screening test results. The other three were diagnosed at the mean age of 7, when they visited the hospital for evaluation of developmental delay and lens dislocation. The group diagnosed at early infancy had normal cognitive function, but the other group had varying degrees of mental retardation. Major complications were found only in the group diagnosed after infancy. CBS gene mutation was found in all the patients, and they were all non-responsive to vitamin B6 treatment. At present, all patients' diets are controlled following a methionine-free formula and they are on medication with folic acid, betaine, pyridoxine, and methylcobalamin. Conclusion: Six homocystinuria patients were monitored for up to 23 years. The group diagnosed at early infancy exhibited no major complications. Therefore, early diagnosis is crucial in the prognosis, and homocystinuria must be included in the newborn screening program.

• 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.

• KSBB Journal
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• v.23 no.5
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• pp.425-430
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• 2008

Hydrogen sulfide ($H_2S$) is a by-product of metabolism of amino acids including sulfur and alcoholic fermentation, it is generally thought of in terms of a poisonous gas. Though $H_2S$ can have a negative impact on the perceived quality of fermented drinks due to an undesirable aroma, it plays prominent roles as a neuromodulator in the mammalian brain as well as a smooth muscle relaxant. Nowadays studies on the proteins which produce $H_2S$ are carried out in various fields such as structure, function, and metabolism. Here we propose to develop a simple and rapid $H_2S$ forming assay method, which will lead to speed up preparing the $H_2S$ forming proteins for identification by MALDI-TOF MS analysis. We detected three kinds of proteins which produce $H_2S$ in the crude extract of Saccharomyces cerevisiae. Those proteins were cystathionie $\beta$-synthase, O-acetylserine sulfhydrylase, and cystathionine $\gamma$-lyase.

• YAKHAK HOEJI
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• v.53 no.2
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• pp.74-77
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• 2009

Food deprivation decreases hepatic glutathione (GSH) levels, which is ascribed to alterations in availability of hepatic cysteine, a rate limiting factor for the GSH synthesis. The present study examines the effects of food deprivation on hepatic metabolism of sulfur amino acid in male rats. In rats fasted for 24 or 48 hours, hepatic GSH levels were decreased from $6.70{\pm}0.16{\mu}mol/g$ liver to $4.02{\pm}0.20$ or $4.06{\pm}0.07{\mu}mol/g$ liver, respectively. Hepatic S-adenosylmethionine levels were also decreased in fasted rats, but S-adenosylhomocysteine levels were increased. Hepatic methionine levels were not changed by food deprivation for 48 hours. On the other hand, hepatic cysteine or taurine levels were increased from $106.2{\pm}4.1$ to $130.0{\pm}2.7$ nmol/g liver or from $2.45{\pm}0.43$ to $5.07{\pm}0.78{\mu}mol/g$ liver, respectively, in 48-hour fasted rats. Activity of cystathionine beta-synthase catalyzed homocysteine to cystathionine, was markedly decreased, but activity of betaine homocysteine methyltransferase was increased in fasted rats, indicating that methylation of homocysteine to methionine is activated. Also activity of cysteine dioxygenase, involved in taurine synthesis, was increased. These results suggested that hepatic methionine levels were maintained in rats fasted for 48 hours through increase in homocysteine methylation, and hepatic GSH may serve as a cysteine supplier reservoir in fasting state.

• 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).