Effect of Mutations of Five Conserved Histidine Residues in the Catalytic Subunit of the cbb3 Cytochrome c Oxidase on its Function

Oh Jeong-Il The Microbiological Society of Korea 2006 The journal of microbiology Vol.44 No.3

The cbb3 cytochrome c oxidase has the dual function as a terminal oxidase and oxygen sensor in the photosynthetic bacterium, Rhodobacter sphaeroides. The cbb3 oxidase forms a signal transduction pathway together with the PrrBA two-component system that controls photosynthesis gene expression in response to changes in oxygen tension in the environment. Under aerobic conditions the cbb3 oxidase generates an inhibitory signal, which shifts the equilibrium of PrrB kinase/phosphatase activities towards the phosphatase mode. Photosynthesis genes are thereby turned off under aerobic conditions. The catalytic subunit (CcoN) of the R. sphaeroides cbb3 oxidase contains five histidine residues (H2l4, B233, H303, H320, and H444) that are conserved in all CcoN subunits of the cbb3 oxidase, but not in the catalytic subunits of other members of copper-heme superfamily oxidases. H214A mutation of CcoN affected neither catalytic activity nor sensory (signaling) function of the cbb3 oxidase, whereas H320A mutation led to almost complete loss of both catalytic activity and sensory function of the cbb3 oxidase. H233V and H444A mutations brought about the partial loss of catalytic activity and sensory function of the cbb3 oxidase. Interestingly, the H303A mutant form of the cbb3 oxidase retains the catalytic function as a cytochrome c oxidase as compared to the wild-type oxidase, while it is defective in signaling function as an oxygen sensor. H303 appears to be implicated in either signal sensing or generation of the inhibitory signal to the PrrBA two-component system.

Effect of Mutations of Five Conserved Histidine Residues in the Catalytic Subunit of the cbb3 Cytochrome c Oxidase on its Function

Jeong-Il Oh 한국미생물학회 2006 The journal of microbiology Vol.44 No.3

The cbb3 cytochrome c oxidase has the dual function as a terminal oxidase and oxygen sensor in the photosynthetic bacterium, Rhodobacter sphaeroides. The cbb3 oxidase forms a signal transduction pathway together with the PrrBA two-component system that controls photosynthesis gene expression in response to changes in oxygen tension in the environment. Under aerobic conditions the cbb3 oxidase generates an inhibitory signal, which shifts the equilibrium of PrrB kinase/phosphatase activities towards the phosphatase mode. Photosynthesis genes are thereby turned off under aerobic conditions. The catalytic subunit (CcoN) of the R. sphaeroides cbb3 oxidase contains five histidine residues (H214, H233, H303, H320, and H444) that are conserved in all CcoN subunits of the cbb3 oxidase, but not in the catalytic subunits of other members of copper-heme superfamily oxidases. H214A mutation of CcoN affected neither catalytic activity nor sensory (signaling) function of the cbb3 oxidase, whereas H320A mutation led to almost complete loss of both catalytic activity and sensory function of the cbb3 oxidase. H233V and H444A mutations brought about the partial loss of catalytic activity and sensory function of the cbb3 oxidase. Interestingly, the H303A mutant form of the cbb3 oxidase retains the catalytic function as a cytochrome c oxidase as compared to the wild-type oxidase, while it is defective in signaling function as an oxygen sensor. H303 appears to be implicated in either signal sensing or generation of the inhibitory signal to the PrrBA two-component system.

오이조직 배양세포에 의한 Ascorbate Oxidase 생성 및 생산

이종화,정호권,新名淳言,임번삼 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.4

오이의 각 기관별로 ascorbate oxidase 활성을 측정한 결과 어린열매(개화 후 4일), 특히 표피부분의 효소활성이 가장 높았으며 total RNA값도 높게 나타났다. Callus 유도에는 LS 기본배지가 최적이었으며 어린열매의 표피부분으로부터 연노랑색의 callus를 얻을 수 있었고, 7개월간의 계대배양한 결과 초기 효소활성의 5배를 갖는 우량 callus cell line을 선발할 수 있었다. 액체진탕배양에 있어 오이 배양세포는 약 25일의 배양기간을 가지며, 세포내 효소활성은 배양초기에 증가하다가 점차 감소하였다. 오이 배양세포는 ascorbate oxidase를 배지로 분비하였으며 세포내 효소활성에 비해 약 4배 정도 높았고 분비량은 배양기간 경과에 따라 증가하였다. 식물성장호르몬으로는 2, 4-D와 kinetin이 효소생산에 적합하였다. Ascorbate oxidase는 CuSO_4 농도에 민감한 반응을 보여, 10 μM 첨가시 LS 기본배지에 비해 약 2배 효소활성이 증가하였다. 배양초기에 간헐적으로 10 μM의 CuSO_4를 첨가한 결과 세포내 효소활성이 급격히 증가하였고 배양 최종일까지 높은 활성을 유지했으나, 배지중에 분비되는 효소활성에는 큰 영향은 없었다. 이상의 결과를 바탕으로 실시한 jar fermentor 배양결과, Erlenmyer flask를 이용한 액체진탕배양과 유사한 경향을 보였으나, 배양기간은 약 16일 정도로 단축되었으며 식물성장호르몬 및 CuSO_4의 첨가조건을 최적화함으로써 배지로 분비되는 효소는 물론 배양세포내의 효소활성을 증가시킬 수 있었다. Ascorbate oxidase activity in various cucumber tissue extracts was highest in young fruit peeling. Cucumber callus was induced from young fruit peeling and callus cell lines were selected for more than 7 months, which produced high levels of ascorbate oxidase and had a high growth rate. Induction of callus was optimized with Linsmaier-Skoog(LS) medium at 25℃ in dark phase. Ascorbate oxidase activity reached a maximum at 5 days after transfer to LS basal liquid-medium and then declined. The enzyme activity in callus cells was stimulated by addition of 10 μM CuSO_4 in the early logarithmic phase of growth. And also, adding 10 μM CuSO_4, at 3rd day and 7th day of culture period, ascorbate oxidase activity in callus cells was maintained to high level. Maximum yield of ascorbate oxidase was found at the 25th day by flask shaking culture, but three-fold of ascorbate oxidase activity was obtained at the 16th day by jar fermentation.

용매별 감잎 추출물의 Xanthine Oxidase 저해효과

문숙희,이민경,채기수 한국식품영양학회 2001 韓國食品營養學會誌 Vol.14 No.2

시중에서 흔하게 구할 수 있고 즐겨 마시는 차로 이용이 되고 있는 감잎을 시료로 택하여 메탄올로 추출한 후 헥산, 클로로포름, 에틸아세테이트, 부탄올, 물 등으로 각각 분획하여 xanthine oxidase에 대한 저해 효과를 살펴보았다. 메탄을 추출물에서는 시료 2.0㎎/ml 첨가 시 78%의 높은 저해효과를 나타내었으며, 농도가 증가할수록 저해효과가 우수한 것으로 나타났다. 또한 메탄올 추출물을 농도별로 첨가하여 반응시간에 따른 저해효과를 살펴본 결과 반응시간 1분에 가장 급속한 저해효과를 나타내었으며 시간이 증가할수록 효소 저해율의 증가는 완만하였다. 한편 각 용매별 획분에서도 마찬가지로 농도가 증가할수록 xanthine oxidase에 대한 저해효과가 증가하였으며 특히 에틸아세테이트 획분에서는 2.0㎎/ml 첨가시 87%의 높은 저해율을 나타내어 xanthine oxidase에 대해 저해 효과가 가장 뛰어남을 알 수 있었다. 각 용매별 획분에 대하여 반응 시간별로 잔존하는 xanthine oxidase의 활성도는 에틸아세테이트 획분과 헥산 획분에서 가장 낮은 반면 물 획분에서 가장 높았으며, 각 획분 모두 반응시간 1분 안에 xanthine oxidase의 활성도가 급격히 감소되었고 그 이후 시간이 경과함에 따라서는 다소 완만한 감소를 나타내었다. The inhibitory effects of xanthine oxidase by the methanol extract and the solvent fractions obtained from persimmon leaves were investigated. The inhibition ratio of xanthine oxidase was 78% by addition of 2.0㎎/ml of the methanol extract. Among the solvent fractions, the ethylacetate fraction showed the strongest inhibitory effect against the xanthine oxidase, followed by the hexane fraction. The effect increased with addition of the ethylacetate fraction. At a concentration of 2.0㎎/ml of the ethylacetate fraction, 65% of the enzyme activity decreased within 1.0 min of incubation with xanthine oxidase. But the activity of xanthine oxidase did not decrease significantly by the length of the incubation time.

토양 미생물로부터 생산된 Extracellular Cholesterol Oxidase의 특성

박정수(Jeong-Su Park),정종문(Jong-Moon Jeong) 한국식품영양과학회 2008 한국식품영양과학회지 Vol.37 No.11

본 연구에서는 산업적으로 사용될 수 있는 안정하고 활성이 높은 cholesterol oxidase를 생산하는 균주를 얻기 위해 토양 미생물로부터 균주를 선별하였다. 선별된 균주에 대하여 세포외 효소 활성을 측정한 결과 BEN 115로 명명한 균주가 가장 높은 효소 활성도를 나타내었다. 이 균주는 형태학적, 생리학적 특성과 배양형태 및 G+C 함량을 분석한 결과 Nocardia속으로 확인되었다. 최적 효소 생산 조건을 조사한 결과 기존 yeast malt extract broth 조성인 0.4% yeast extract, 0.4% glucose, 1% malt extract에서 가장 높은 활성을 나타냈다. 본 균주에서 생산된 extracellular cholesterol oxidase는 SDS-PAGE와 Western blot 결과 분자량이 55, 57 kDa인 두 종류의 효소가 존재하는 것으로 나타났다. BEN 115에 대한 효소학적 특성을 연구한 결과 온도 안정성은 55℃까지 효소 활성이 유지되었고, pH 안정성은 pH 3.5~9.5의 범위까지 안정한 것으로 나타났으며 최적 온도와 최적 pH는 각각 35oC와 pH 5.5인 것으로 나타났다. 또한 detergent(Triton X-100, Triton X-114 그리고 Tween 80) 첨가 시 효소 활성이 첨가하지 않은 대조군보다 약 1.6~2.0배 증가하는 것으로 나타났다. Campesterol, sitosterol 그리고 stigmasterol에 대한 기질 특이성은 cholesterol(100%)과 상대적으로 비교 시 각각 50%, 50% 그리고 27%의 기질 특이성을 나타내었다. Cholesterol oxidase catalyses the conversion of cholesterol to 4-cholesten-3-one. This enzyme has been used for clinical assay of human serum cholesterol and for reduction of cholesterol level in foods and feeds. In order to search the microorganism which has a high extracellular and stable activity of cholesterol oxidase, soil microorganisms were screened. As a result, the one with the highest extracellular cholesterol oxidase activity was obtained and named as the BEN 115. The BEN 115 strain was identified as one of the Nocardia species based on our taxonomic studies. The cholesterol oxidase from this strain was shown to have two bands of extracellular proteins on SDS-PAGE and Western blot. Their molecular masses were estimated to be about 55 and 57 kDa, respectively. In addition, this cholesterol oxidase was considerably stable at the broad range of pH 3.5~9.5 and at the temperature of 25~55℃. The optimum pH and temperature of this cholesterol oxidase were pH 5.5 and 35℃, respectively. The activity of extracellular cholesterol oxidase could be enhanced 1.6 to 2.0 folds by the addition of nonionic detergent such as Triton X-114, Triton X-100, or Tween-80 into the culturing broth. The substrate specificities against campesterol, sitosterol and stigmasterol were measured to be 50%, 50%, and 27%, respectively, compared to the cholesterol. These results suggest that Nocardia sp. BEN 115 may be useful as a microbial source of cholesterol oxidase production.

Allopurinol modulates reactive oxygen species generation and Ca²⁺ overload in ischemia-reperfused heart and hypoxia-reoxygenated cardiomyocytes

Kang, Seok-Min,Lim, Soyeon,Song, Heesang,Chang, Woochul,Lee, Sunju,Bae, Sang-mee,Chung, Ji Hyung,Lee, Hakbae,Kim, Ho-Gyoung,Yoon, Deok-Hyo,Kim, Tae Woong,Jang, Yangsoo,Sung, Jae-Mo,Chung, Nam-Sik,Hwan Elsevier 2006 european journal of pharmacology Vol.535 No.1

<P><B>Abstract</B></P><P>Myocardial oxidative stress and Ca<SUP>2+</SUP> overload induced by ischemia-reperfusion may be involved in the development and progression of myocardial dysfunction in heart failure. Xanthine oxidase, which is capable of producing reactive oxygen species, is considered as a culprit regarding ischemia-reperfusion injury of cardiomyocytes. Even though inhibition of xanthine oxidase by allopurinol in failing hearts improves cardiac performance, the regulatory mechanisms are not known in detail. We therefore hypothesized that allopurinol may prevent the xanthine oxidase-induced reactive oxygen species production and Ca<SUP>2+</SUP> overload, leading to decreased calcium-responsive signaling in myocardial dysfunction. Allopurinol reversed the increased xanthine oxidase activity in ischemia-reperfusion injury of neonatal rat hearts. Hypoxia-reoxygenation injury, which simulates ischemia-reperfusion injury, of neonatal rat cardiomyocytes resulted in activation of xanthine oxidase relative to that of the control, indicating that intracellular xanthine oxidase exists in neonatal rat cardiomyocytes and that hypoxia-reoxygenation induces xanthine oxidase activity. Allopurinol (10 μM) treatment suppressed xanthine oxidase activity induced by hypoxia-reoxygenation injury and the production of reactive oxygen species. Allopurinol also decreased the concentration of intracellular Ca<SUP>2+</SUP> increased by enhanced xanthine oxidase activity. Enhanced xanthine oxidase activity resulted in decreased expression of protein kinase C and sarcoendoplasmic reticulum calcium ATPase and increased the phosphorylation of extracellular signal-regulated protein kinase and p38 kinase. Xanthine oxidase activity was increased in both ischemia-reperfusion-injured rat hearts and hypoxia-reoxygenation-injured cardiomyocytes, leading to reactive oxygen species production and intracellular Ca<SUP>2+</SUP> overload through mechanisms involving p38 kinase and extracellular signal-regulated protein kinase (ERK) via sarcoendoplasmic reticulum calcium ATPase (SERCA) and protein kinase C (PKC). Xanthine oxidase inhibition with allopurinol modulates reactive oxygen species production and intracellular Ca<SUP>2+</SUP> overload in hypoxia-reoxygenation-injured neonatal rat cardiomyocytes.</P>

Analysis of Relationship Between Spermatozoa Ability and Reactive Oxygen Species in Porcine: I. Sperm Preincubation by Xanthine and Xanthine Oxidase

박춘근,정희태,김종흥,이상찬,양부근,김정익,Park, C.K.,Cheong, H.T.,Kim, J.H.,Lee, S.C.,Yang, B.K.,Kim, C.I. The Korean Society for Reproductive Medicine 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.3

The objective of this study was to test the effect of catalase on penetration in vitro by spermatozoa preincubated with xanthine and/or xanthine oxidase. The penetration rates were, significantly (p<0.05) higher in spermatozoa preincubated without (66 and 38%) than with (40 and 15%) catalase for 0 and 30 min. When spermatozoa were preincubated and inseminated in medium with xanthine, the penetration rates were significantly higher (p<0.05) in medium with (68, 70 and 49% for 0, 30 and 60 min) than without (33, 41 and 19% for 0, 30 and 60 min) catalase. However, in oocytes were' inseminated with spermatozoa pre incubated with or without catalase in the presence of xanthine oxidase, no decrease in penetrations rates were observed for up to 60 min of preincubation. In another experiment, the penetration rates were significantly (p<0.00l) higher in medium with (75, 55 and 52%) than without (14, 4 and 8%) catalase when oocytes were inseminated with spermatozoa preincubated for 0, 30 and 60 min in the presence of xanthine plus xanthine oxidase. On the other hand, The rate of polyspermy in oocytes penetrated in medium without catalase in the presence of xanthine or xanthine plus xanthine oxidase decreased with time of spermatozoa preincubation. However, no differences were observed in polyspermy rates in the medium with xanthine oxidase alone despite presence of catalase. These results indicate the advantages of spermatozoa pre incubated with xanthine plus xanthine oxidase in the presence of catalase to increase penetration potential and with suppressed polyspermy in porcine. 본 연구는 xanthine과 xanthine oxidase가 첨가된 배양액내에서 전배양된 돼지동결정액의 수정능력에 있어서 catalase의 영향을 검토하였다. 체외수정을 위한 기본 배양액내에서 0 또는 30분간 전배양한 정자는 catalase를 첨가 (40 및 15%)한 경우 보다는 무첨가 (66 및 38%)시 유의적으로 높은 정자침입율을 나타냈지만 (p<0.05), 배양액내에 xanthine을 첨가해 정자를 0, 30 및 60분간 전배양했을 때에는 catalase 무첨가 (33, 41 및 19%) 보다는 첨가시 (68, 70 및 49%) 유의적으로 높은 정자침입율이 인정되었다 (p<0.05). 그러나 xanthine oxidase를 첨가하여 정자의 전배양을 행하지 않은 경우는 catalase의 첨가 (13%) 보다는 무첨가 (51%)시 유의적으로 높은 정자침입율을 나타냈지만 (p<0.01), 전배양(30 및 60분)후에는 catalase의 존재유무와 정자전배양시간에 관계없이 매우 낮은 정자침입율 $(10\sim21%)$을 나타냈다. xanthine과 xanthine oxidase를 동시에 첨가하여 0, 30 및 60분간 정자를 전배양한 경우 catalase의 무첨가 (14, 4 및 8%)보다 첨가 (75, 55 및 52%)시 유의적으로 높은 정자침입율을 나타냈다 (p<0.001). 한편, 다정자침입율은 xanthine, xanthine+xanthine oxidase의 첨가시 정자전배양기간이 길어짐에 따라 감소하였으며, catalase의 첨가보다는 무첨가시 낮은 다정자침입율을 나타냈다. 본 연구의 결과로부터 xanthine과 xanthine oxidase를 동시에 첨가시 catalase의 존재는 정자의 전배양후에도 다정자침입을 억제하면서 수정능력의 유지를 위해 매우 효과적인 것으로 추측되었다.

감잎 열탕 추출물 및 감잎 탄닌의 Xanthine Oxidase 저해 효과

문숙희,이민경 한국식품영양학회 1998 韓國食品營養學會誌 Vol.11 No.3

우리 나라에서 쉽게 구할 수 있고, 값이 저렴하여 대중적으로 널리 이용될 수 있는 감잎으로부터의 열탕 추출물과 함께 탄닌을 분리하여 xanthine oxidase 활성을 측정함으로써 감잎의 통풍 예방효과를 살펴보았다. 감잎의 열탕 추출물을 농도별로 첨가하여 xanthine oxidase 저해효과를 살펴본 결과 열탕 추출물의 농도가 증가할수록 저해효과가 증가하여 20mg 첨가시 82.4%의 높은 저해효과를 나타냈으며, 감잎탄닌도 감잎의 열탕 추출물과 마찬가지로 농도에 비례해서 xanthine oxidase 저해효과가 증가하는 것으로 나타났다. 한편 감잎 열탕추출물 및 감잎 탄닌은 xanthine oxidase에 대한 경쟁적 저해를 하는 것으로 나타났다. The influence of hot water extracts and tannin obtained from persimmon leaves on xanthine oxidase were investigated. Above two samples had higher inhibitory effects against xanthine oxidase, and the effects were increased with addition of the samples. The inhibition ratio of xanthine oxidase by hot water extracts and tannin obtained from persimmon leaves was 92.4% and 92.1% by addition of 2.0mg/ml of the hot water extracts and the tannin, respectively. The inhibitions by the hot water extracts and the tannin were of competitive mode with respect to xanthine as a substrate.

Chemical Modification of Residue of Lysine, Tryptophan, and Cysteine in Spinach Glycolate Oxidase

Lee, Duk-Gun,Cho, Nam-Jeong,Choi, Jung-Do Korean Society for Biochemistry and Molecular Biol 1996 Journal of biochemistry and molecular biology Vol.29 No.4

Spinach glycolate oxidase was subjected to a series of chemical modifications aimed at identifying amino acid residues essential for catalytic activity. The oxidase was reversibly inactivated by treatment with pyridoxal 5'-phosphate (PLP). The inactivation by PLP was accompanied by the appearance of an absorption peak of around 430 nm, which was shifted to 325 nm upon reduction with $NaBH_4$. After reduction, the PLP-treated oxidase showed a fluorescence spectrum with a maximum of around 395 nm by exciting at 325 nm. The substrate-competitive inhibitors oxalate and oxaloacetate provided protection against inactivation of the oxidase by PLP. These results suggest that PLP inactivates the enzyme by fonning a Schiff base with lysyl residue(s) at an active site of the oxidase. The enzyme was also inactivated by tryptophan-specific reagent N-bromosuccinimide (NBS). However, competitive inhibitors oxalate and oxaloacetate could not protect the oxidase significantly against inactivation of the enzyme by NBS. The results implicate that the inactivation of the oxidase by NBS is not directly related to modification of the tryptophanyl residue at an active site of the enzyme. Treatments of the oxidase with cysteine-specific reagents iodoacetate, silver nitrate, and 5,5'-dithiobis-2-nitrobenzoic acid did not affect significantly the activity of the enzyme.

항통풍성 Xanthin oxidase저해물질 생산 야생효모 Meyerozyma guilliermondii SI 10-3의 균학적 특성과 Xanthin oxidase 저해물질의 생산

박선정, 박혜영, 심혜령, 이종수 배재대학교 자연과학연구소 2020 自然科學論文集 Vol.31 No.1

This study was carried out to produce novel anti-gout xanthin oxidase(XOD) inhibitor from non-pathogenic wild yeast obtained from soils of Daechung dam of Shintanjin, Daejeon city, Korea. We finally selected Meyerozyma guilliermondii SI 10-3 which showed the highest 29.5% of XOD inhibitory activity. Microbiological characteristics of Meyerozyma guilliermondii SI 10-3 was investigated. Meyerozyma guilliermondii SI 10-3 was global in shape with 1.4 x 1.4 ㎛, and not formed ascospore and pseudomycelium. Meyerozyma guilliermondii SI 10-3 strain grew well in YPD, YM and PD media, but not grew in vitamin-free medium. It was also halophilic strain which was grew in 5% NaCl-containing YPD medium. Maximal production of XOD inhibitor was obtained when Meyerozyma guilliermondii SI 10-3 was cultured at 30℃ for 36h in YPD medium and its inhibitory activity was showed 44.8%. 항통풍성 xanthin oxidase 저해활성이 우수한 비병원성 야생효모를 선발하여 산업적으로 응용할 목적으로 대전광역시 신탄진소재 대청댐주변 토양과 부식물등에서 분리, 동정한 비병원성 야생효모들의 무세포 추출물들을 제조하여 이들의 xanthin oxidase 저해활성을 조사하여 우수 효모들을 선발하였다. 이들중, 대전광역시 3대 하천에서 분리한 Meyerozyma guilliermondii SI 10-3이 가장높은 29.5%의 xanthin oxidase 저해활성을 보여 최종 선발하였다. 선발균주는 구형으로 분열법으로 무성생식하였고 포자와 의균사를 형성하지 않았다. yeast extract-peptone-dextrose (YPD) 배지와 yeast extract-malt extract 배지에서 잘 생육하였으나 비타민을 첨가하지 않은 YPD 배지에서는 생육하지 못했다. 또한, 20% 포도당과 5% NaCl을 각각 함유한 YPD 배지에서 생육하는 내당성과 호염성을 보였다. Meyerozyma guilliermondii SI 10-3의 xanthin oxidase저해활성은 선발균주를 30℃에서 36시간동안 YPD 배지에서 배양하였을 때 44.8%의 최고 저해활성을 나타냈다.