인간 Cystathionine γ-Lyase에 의한 Selenomethionine의 Methylselenol로의 직접분해

조현남 ( Hyun Nam Cho ),지광환 ( Kwang Hwan Jhee ) 한국미생물생명공학회(구 한국산업미생물학회) 2014 한국미생물·생명공학회지 Vol.42 No.1

Selenium(Se)은 사람에게 필수성분이지만 독성이 강한 물질이다. 따라서 생체에서 Se(셀레늄)의 농도조절은 정확하고 효율적으로 이루어져야 한다. 사람은 Se을 음식의 형태로 섭취해야 하고 주로 seleno-L-methionine (L-SeMet)의 형태로 섭취한다. 섭취된 L-SeMet은 methionine 대사와 동일한 효소를 이용하여 Se-adenosyl-L-SeMet으로 대사된다고 알려져 있었다. 그러나 최근 쥐의 간 추출물의 실험에서, L-SeMet이 cystathionine γ-lyase (mouse CGL)의 작용으로 methylselenol(CH3SeH)로 직접적으로 대사된다는 보고가 있었다. CGL은 원래 cystathionine을 L-cysteine과 α-ketobutyrate, 그리고 NH3로 분해하는 효소로 알려져 있었다. 따라서 본 연구에서는 쥐의 간 추출물 대신에 인간의 CGL을 분리 정제하여 LSeMet에서 methylselenol의 형성을 확인하고자 하였다. Methylselenol의 표준시료는 dimethyldiselenide를 sodium tetrahydroborate로 환원시켜 준비하였다. 그리고 L-SeMet을 기질로 사용한 효소 반응액 중에서 가스상의 생성물은 GC/MS 스펙트럼으로 분석하였다. 분석 결과 methylselenol의 유도체인 dinitrophenyl selenoether와 일치하였다. 또한 인간 CGL이 L-SeMet에서 methylselenol을 형성하는 반응의 kinetic parameter를 mouse CGL과 비교 분석하였다. 결과적으로 human CGL은 섭취된 L-SeMet의 대사를 책임지고 있는 중요한 효소이다. 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 (CH3SeH) in mouse liver extract by the reaction of cystathionine γ-lyase (CGL). The canonical reaction of CGL was known to catalyze the cleavage of L-cystathionine to L-cysteine, α-ketobutyrate and NH3. In the present study, we found that L-SeMet could be directly converted to CH3SeH using purified homogenous human CGL instead of mouse liver cytosol. Authentic CH3SeH 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 CH3SeH 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.

새로운 신경전달물질 H₂S 발생 효소, cystathionine γ-lyase의 대량발현 조건과 활성측정

Kyoung-Ran Kim(김경란),Hae-Jung Byun(변혜정),Hyun-Nam Cho(조현남),Jung-Hyun Kim(김정현),Seun-Ah Yang(양선아),Kwang-Hwan Jhee(지광환) 한국생명과학회 2011 생명과학회지 Vol.21 No.1

질병과 밀접한 관계가 있는 hCGL 단백질의 경우 대량 배양 시 유도체를 사용하지 않아도 발현이 되는 점과 유전자 측면에서 조작이 쉬운 E.coli를 이용하여도 발현이 된다는 점에 있어서 중요한 이점을 가지고 있다. 본 연구에서는 배양되는 온도와 발현에 관련 있는 유도체의 농도, 600 ㎚에서의 균 성장 정도에 따른 유도체의 첨가 그리고 배지의 양을 조절하면서 유입되는 aeration의 조건으로 hCGL 단백질 발현의 최적의 조건 확립을 목적으로 하였다. 또 각 발생되는 inclusion body의 양을 측정하면서 보다 많은 가용성 단백질을 발현시키는 조건을 확립하고자 하였다. hCGL 단백질은 저온에서 보다 많은 양의 단백질이 발현되며 inhibitor의 억제를 담당하는 유도체의 농도와는 상관없이 발현이 되었다. 또한 균의 성장 정도에 따라 유도체의 첨가시기를 달리 하였을 때, 발현 비율에 차이는 있었으나 전체적인 단백질 양과 비교해 보면, 이는 hCGL 발현에 큰 영향을 미치지 않는다. 배지의 양을 달리하여 살펴본 aeration에 따른 hCGL 발현 정도는 배지의 부피가 15%일 때 높은 aeration으로 균의 양은 많았으나 목적 단백질인 hCGL의 발현은 aeration이 되지 않는 조건에서 더 잘되는 것을 확인하였다. 그리고 His-TEV-hCGL의 활성은 야생형 hCGL의 활성을 기준으로 하였을 때, L-cystathionine을 기질로 하였을 경우 76%, L-cysteine을 기질로 하였을 경우 88% 수준으로 유사한 활성을 나타내었고, 이는 손쉽게 정제 가능한 His-TEV-hCGL을 야생형을 대신하여 사용할 수 있음을 시사한다. 또한 His-TEV-hCGL이 야생형 hCGL과 같이, 427 ㎚에서 흡광을 가지는 것으로 보아 보효소PLP를 포함하고 있음을 알 수 있었다. 이로써 homocysteine 대사연구에 필수적인 hCGL 효소를 다량 얻는 방법을 확립하고, 관련 연구에 기여하리라 사료된다. There is a growing recognition of the significance of H2S as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL), are believed to be chiefly responsible for H₂S 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 α-γ elimination reaction with L-cystathionine and 88% for α-β elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 ㎚ absorption maximum, which is attributed to the binding cofactor, pyridoxal 5’-phosphate (PLP).

Hydrogen sulfide-producing cystathionine γ-lyase is critical in the progression of kidney fibrosis

Han, Sang Jun,Noh, Mi Ra,Jung, Jung-Min,Ishii, Isao,Yoo, Jeongsoo,Kim, Jee In,Park, Kwon Moo Elsevier 2017 FREE RADICAL BIOLOGY AND MEDICINE Vol.112 No.-

<P><B>Abstract</B></P> <P>Cystathionine γ-lyase (CSE), the last key enzyme of the transsulfuration pathway, is involved in the production of hydrogen sulfide (H<SUB>2</SUB>S) and glutathione (GSH), which regulate redox balance and act as important antioxidant molecules. Impairment of the H<SUB>2</SUB>S- and GSH-mediated antioxidant system is associated with the progression of chronic kidney disease (CKD), characterized by kidney fibrosis and dysfunction. Here, we evaluated the role of CSE in the progression of kidney fibrosis after unilateral ureteral obstruction (UUO) using mice deficient in the <I>Cse</I> gene. UUO of wild-type mice reduced the expression of H<SUB>2</SUB>S-producing enzymes, CSE, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase in the obstructed kidneys, resulting in decreased H<SUB>2</SUB>S and GSH levels. <I>Cse</I> gene deletion lowered H<SUB>2</SUB>S and GSH levels in the kidneys. Deleting the <I>Cse</I> gene exacerbated the decrease in H<SUB>2</SUB>S and GSH levels and increase in superoxide formation and oxidative damage to proteins, lipids, and DNA in the kidneys after UUO, which were accompanied by greater kidney fibrosis, deposition of extracellular matrixes, expression of α-smooth muscle actin, tubular damage, and infiltration of inflammatory cells. Furthermore, <I>Cse</I> gene deletion exacerbated mitochondrial fragmentation and apoptosis of renal tubule cells after UUO. The data provided herein constitute in vivo evidence that <I>Cse</I> deficiency impairs renal the H<SUB>2</SUB>S- and GSH-producing activity and exacerbates UUO-induced kidney fibrosis. These data propose a novel therapeutic approach against CKD by regulating CSE and the transsulfuration pathway.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Ureteral obstruction (UO) impairs H<SUB>2</SUB>S-producing enzymes, CSE, CBS, and 3-MST. </LI> <LI> UO reduces levels of H<SUB>2</SUB>S and GSH in the kidney. </LI> <LI> CSE deletion reduces H<SUB>2</SUB>S and GSH and exacerbates UO-induced those in the kidney. </LI> <LI> CSE deletion worsens oxidative injury and apoptosis, resulting in fibrosis. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Regulation of Enzymes Involved in Methionine Biosynthesis in Corynebacterium glutamicum

( Yeom Hye Jin ),( Byung Joon Hwang ),( Myong Sok Lee ),( Youn Hee Kim ),( Heung Shick Lee ) 한국미생물생명공학회 2004 Journal of microbiology and biotechnology Vol.14 No.2

Beneficial Role of Hydrogen Sulfide in Renal Ischemia Reperfusion Injury in Rats

최은경,박솔희,임정아,홍성욱,곽경화,박성식,임동건,정훈 연세대학교의과대학 2018 Yonsei medical journal Vol.59 No.8

Purpose: Hydrogen sulfide (H2S) is an endogenous gaseous molecule with important physiological roles. It is synthesized fromcysteine by cystathionine γ-lyase (CGL) and cystathionine β-synthase (CBS). The present study examined the benefits of exogenousH2S on renal ischemia reperfusion (IR) injury, as well as the effects of CGL or CBS inhibition. Furthermore, we elucidatedthe mechanism underlying the action of H2S in the kidneys. Materials and Methods: Thirty male Sprague-Dawley rats were randomly assigned to five groups: a sham, renal IR control, sodiumhydrosulfide (NaHS) treatment, H2S donor, and CGL or CBS inhibitor administration group. Levels of blood urea nitrogen(BUN), serum creatinine (Cr), renal tissue malondialdehyde (MDA), and superoxide dismutase (SOD) were estimated. Histologicalchanges, apoptosis, and expression of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulatedkinase, c-Jun N-terminal kinase, and p38) were also evaluated. Results: NaHS attenuated serum BUN and Cr levels, as well as histological damage caused by renal IR injury. Administration ofNaHS also reduced oxidative stress as evident from decreased MDA, preserved SOD, and reduced apoptotic cells. Additionally,NaHS prevented renal IR-induced MAPK phosphorylation. The CGL or CBS group showed increased MAPK family activity; however,there was no significant difference in the IR control group. Conclusion: Exogenous H2S can mitigate IR injury-led renal damage. The proposed beneficial effect of H2S is, in part, because ofthe anti-oxidative stress associated with modulation of the MAPK signaling pathways.

Hypothalamic-Pituitary Axis Regulates Hydrogen Sulfide Production

Hine, Christopher,Kim, Hyo-Jeong,Zhu, Yan,Harputlugil, Eylul,Longchamp, Alban,Matos, Marina Souza,Ramadoss, Preeti,Bauerle, Kevin,Brace, Lear,Asara, John M.,Ozaki, C. Keith,Cheng, Sheue-yann,Singha, S Cell Press 2017 Cell metabolism Vol.25 No.6

<▼1><P><B>Summary</B></P><P>Decreased growth hormone (GH) and thyroid hormone (TH) signaling are associated with longevity and metabolic fitness. The mechanisms underlying these benefits are poorly understood, but may overlap with those of dietary restriction (DR), which imparts similar benefits. Recently we discovered that hydrogen sulfide (H<SUB>2</SUB>S) is increased upon DR and plays an essential role in mediating DR benefits across evolutionary boundaries. Here we found increased hepatic H<SUB>2</SUB>S production in long-lived mouse strains of reduced GH and/or TH action, and in a cell-autonomous manner upon serum withdrawal in vitro. Negative regulation of hepatic H<SUB>2</SUB>S production by GH and TH was additive and occurred via distinct mechanisms, namely direct transcriptional repression of the H<SUB>2</SUB>S-producing enzyme cystathionine γ-lyase (CGL) by TH, and substrate-level control of H<SUB>2</SUB>S production by GH. Mice lacking CGL failed to downregulate systemic T<SUB>4</SUB> metabolism and circulating IGF-1, revealing an essential role for H<SUB>2</SUB>S in the regulation of key longevity-associated hormones.</P></▼1><▼2><P><B>Highlights</B></P><P>•<P>Hepatic H<SUB>2</SUB>S production capacity is elevated in long-lived hypopituitary mouse models</P>•<P>Growth hormone (GH) represses hepatic H<SUB>2</SUB>S production post-transcriptionally</P>•<P>Thyroid hormone (TH) acts via TRβ to repress cystathionine γ-lyase and H<SUB>2</SUB>S levels</P>•<P>H<SUB>2</SUB>S negatively regulates circulating TH and IGF-1 levels</P></P></▼2><▼3><P>Reduced thyroid hormone (TH) and growth hormone (GH) activity are hallmarks of genetic models of longevity in mice. Here, Hine et al. find that TH and GH negatively regulate hepatic production of the longevity-associated gas hydrogen sulfide, which feeds back to negatively regulate circulating TH and IGF-1 levels.</P></▼3>

Production of Cephalosporin C Using Crude Glycerol in Fed-Batch Culture of Acremonium chrysogenum M35

신현용,이진영,최한석,이자현,김승욱 한국미생물학회 2011 The journal of microbiology Vol.49 No.5

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.

Simple and Novel Assay of the Host-Guest Complexation of Homocysteine with Cucurbit[7]uril

( Se-ho Park ),( Jae-yeul Lee ),( Hyun-nam Cho ),( Kyoung-ran Kim ),( Seun-ah Yang ),( Hee-joon Kim ),( Kwang-hwan Jhee ) 한국미생물 · 생명공학회 2019 Journal of microbiology and biotechnology Vol.29 No.1

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman’s reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH2CH2SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman’s protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.