• 제목/요약/키워드: Pyridoxal-5'-phosphate

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A Stereochemical Aspect of Pyridoxal 5' -Phosphate Dependent Enzyme Reactions and Molecular Evolution

  • Jhee, Kwang-Hwan;Tohru, Yoshimura;Yoichi, Kurokawa;Nobuyoshi, Esaki;Kenji, Soda
    • Journal of Microbiology and Biotechnology
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    • 제9권6호
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    • pp.695-703
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    • 1999
  • We have studied the stereospecificities of various pyridoxal 5'-phosphate (PLP) dependent enzymes for the hydrogen transfer between the C-4' of a bound coenzyme and the C-2 of a substrate in the transamination catalyzed by the enzymes. Stereospecificities reflect the structures of enzyme active-sites, in particular the geometrical relationship between the coenzyme-substrate Schiff base and the active site base participating in an $\alpha$-hydrogen abstraction. The PLP enzymes studied so far catalyze only a si-face specific (pro-S) hydrogen transfer. This stereochemical finding suggests that the PLP enzymes have the same topological active-site structures, and that the PLP enzymes have evolved divergently from a common ancestral protein. However, we found that o-amino acid aminotransferase, branched chain L-amino acid aminotransferase, and 4-amino-4-deoxychorismate lyase, which have significant sequence homology with one another, catalyze a re-face specific (pro-R) hydrogen transfer. We also showed that PLP-dependent amino acid racemases, which have no sequence homology with any aminotransferases, catalyze a non-stereospecific hydrogen transfer: the hydrogen transfer occurs on both faces of the planar intermediate. Crystallographical studies have shown that the catalytic base is situated on the re-face of the C-4' of the bound coenzyme in o-amino acid aminotransferase and branched chain L-amino acid aminotransferase, whereas the catalytic base is situated on the si-face in other aminotransferases (such as L-aspartate aminotransferase) catalyzing the si-face hydrogen transfer. Thus, we have clarified the stereospecificities of PLP enzymes in relation with the primary structures and three-dimensional structures of the enzymes. The characteristic stereospecificities of these enzymes for the hydrogen transfer suggest the convergent evolution of PLP enzymes.

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

  • Lee, Duk-Gun;Cho, Nam-Jeong;Choi, Jung-Do
    • BMB Reports
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    • 제29권4호
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    • pp.321-326
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    • 1996
  • 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.

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Evaluation of Vitamin $B_{6}$ Status and Korean RDA in Korean College Students Following a Uncontrolled Diet

  • Oho, Youn-Ok;Kim, Young-Nam
    • Nutritional Sciences
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    • 제5권1호
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    • pp.20-25
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    • 2002
  • The vitamin $B_{6}$ status of 49 healthy college student (women, aged 20-26 y) was estimated for evaluation of vitamin $B_{6}$ status and the Korean Recommended Dietary Allowance (RDA) for vitamin $B_{6}$. The average daily vitamin $B_{6}$ intake of the subjects was 0.86 $\pm$ 0.289 mg/d or 61.43 $\pm$ 24.10% of Korean RDA. The average ratio of vitamin $B_{6}$ intake to daily protein intake was 0.014 $\pm$ 0.003 mg/g protein. Foods from animal and plaint sources provided 34.25 $\pm$ 18.62% and 65.78 $\pm$ 18.72%, respectively, of total vitamin $B_{6}$. Plasma pyridoxal 5'-phosphate (PLP) concentration was significantly (p<.01 - p<.001) positively correlated to intakes of all other nutrients except vitamin C. However, no significant correlation was found between plasma PLP and nutrient intake. Vitamin $B_{6}$ intake only tended to have a positive correlation with plasma PLP concentration. Plasma total cholesterol was correlated to plasma PLP concentration (p<.05). Plasma PLP had no correlation with levels of glucose, triglyceride, and albumin. These results confirm that the present Korea RDA for vitamin $B_{6}$ of 1.4mg/d based on 0.02 mg/g protein is adequate.

Vitamin B6 Deficiency, Genome Instability and Cancer

  • Wu, Xia-Yu;Lu, Lin
    • Asian Pacific Journal of Cancer Prevention
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    • 제13권11호
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    • pp.5333-5338
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    • 2012
  • Vitamin B6 functions as a coenzyme in >140 enzymatic reactions involved in the metabolism of amino acids, carbohydrates, neurotransmitters, and lipids. It comprises a group of three related 3-hydroxy-2-methyl-pyrimidine derivatives: pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their phosphorylated derivatives [pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP)], In the folate metabolism pathway, PLP is a cofactor for the mitochondrial and cytoplasmic isozymes of serine hydroxymethyltransferase (SHMT2 and SHMT1), the P-protein of the glycine cleavage system, cystathionine ${\beta}$-synthase (CBS) and ${\gamma}$-cystathionase, and betaine hydroxymethyltransferase (BHMT), all of which contribute to homocysteine metabolism either through folate-mediated one-carbon metabolism or the transsulfuration pathway. Folate cofactors carry and chemically activate single carbons for the synthesis of purines, thymidylate and methionine. So the evidence indicates that vitamin B6 plays an important role in maintenance of the genome, epigenetic stability and homocysteine metabolism. This article focuses on studies of strand breaks, micronuclei, or chromosomal aberrations regarding protective effects of vitamin B6, and probes whether it is folate-mediated one-carbon metabolism or the transsulfuration pathway for vitamin B6 which plays critical roles in prevention of cancer and cardiovascular disease.

Characterization of a Gene Encoding Diaminopimelate Decarboxylase from Rice

  • Kim, Jung-Sup;Lee, Soon-Dong
    • Animal cells and systems
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    • 제10권4호
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    • pp.197-201
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    • 2006
  • Diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) catalyzes the conversion of diaminopimelate into lysine (Lys), which is the last step in Lys biosynthetic pathway. The genes for DAPDC have been reported in many bacteria, and more recently in Arabidopsis. Here we report characterization of a gene for DAPDC from rice (OsDAPDC). Sequence analysis of a cDNA clone revealed a full-length open reading frame for OsDAPDC that encoded 490 amino acids, approximately 53.2 kDa protein. The OsDAPDC protein contains a consensus binding site for pyridoxal-5'-phosphate as a cofactor and has a sequence at the amino terminus that resembles a transit peptide for localization to plastids, similar to that of Arabidopsis. Single gene encoding DAPDC was found in chromosome II in rice. The predicted amino acid sequence of OsDAPDC is highly homologous to that of the enzymes for DAPDC encoded by lysA of many bacteria. Expression of OsDAPDC in lysA mutants of Escherichia coli shows that the gene is able to functionally complement the mutants. These results suggest that OsDAPDC encodes a protein for diaminopimelate decarboxylase in rice.

Inactivation of Brain myo-Inositol Monophosphate Phosphatase by Pyridoxal-5'-Phosphate

  • Kim, Dae-Won;Hong, Joung-Woo;Eum, Won-Sik;Choi, Hee-Soon;Choi, Soo-Hyun;Kim, So-Young;Lee, Byung-Ryong;An, Jae-Jin;Lee, Sun-Hwa;Lee, Seung-Ree;Kwon, Oh-Shin;Kwon, Hyeok-Yil;Cho, Sung-Woo;Lee, Kil-Soo;Park, Jin-Seu;Choi, Soo-Young
    • BMB Reports
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    • 제38권1호
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    • pp.58-64
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    • 2005
  • Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

ENZYMATIC STUDIES ON VITAMIN B6 METABOLISM

  • Kim, Young-Tae
    • 한국어병학회지
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    • 제6권2호
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    • pp.133-142
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    • 1993
  • 비타민은 동물과 어류에 있어 성장, 발육, 및 대사기능에 비교적 적은 양으로 요구되는 필수 미량원소이다. 이들 미세 영양분이 결핍됨으로써 식욕 감퇴에서부터 심한 조직 변형등의 증상들을 일으킨다. 수용성 vitamin B6는 비교적 적은양이 요구되며, 주로 보조 효소 기능을 가지고 있다. 비타민 B6라는 명칭은 유사한 대사 기능들을 가진 화학적으로 관련된 pyridoxine, pyridoxamine, pyridoxal들을 의미한다. 비타민 B6는 비 반추 포유류, 조류와 어류의 음식물을 통해 섭취되는 성분이며, 비타민 B6 화합물들은 식물과 미생물 등에 의해 합성된다. 대사적으로 활성형인 비타민 B6 보조효소는 pyridoxal-5-phsphate(PLP)이며, decarboxylases, aminotransferases, sulfhydrases, tryptophanases를 포함한 아미노산 대사에 관여하는 여러 효소들(PLP-dependant)에 coenzyme으로 작용한다. 비타민 B6 요구량이 육식 동물보다 고단백질을 섭취하는 어류에서 더 높다. B6는 탄수화물과 지질의 대사에도 역시 관여하며 heme과 serotonin의 합성에 필수적이다. 어류에서 결핍증(현상)은 빨리 나타나는데, 이러한 증세로는 신경계 분열, 경련, 유영 부조화, 피부병변, 부종, 안구 돌출, 근 긴장성 경련 등이 포함된다. 비타민 B6는 pyridoxal kinase와 pyridoxine(pyridoxamine) oxidase의 촉매 반응에 의해 생체내 활성형인 PLP로 된다. PLP는 PLP-의존성 효소에서 보조 효소로 필수적인 역할을 하는 관계로 이 논문에서는 비타민 B6 생합성 및 대사와 비타민 B6 의존성 효소(aminotransferase)의 특성과 작용기작에 대한 효소학적 연구를 중점으로 이들 enzyme들의 구조와 기능에 대한 최근 연구 동향을 살펴보고자 한다.

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Aspergillus oryzae에 있어서 L-Tyrosine의 분해효소에 관한 연구 (Studies on the Degradation of L-Tyrosine by Aspergillus oryzae)

  • 정동효;박성오;김영진
    • Applied Biological Chemistry
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    • 제14권2호
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    • pp.131-135
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    • 1971
  • 1. Aspergillus oryzae의 균체에는 L-tyrosine-${\alpha}$-ketoglutaric acid transaminase와 p-hydroxyphenlypyruvic acid oxidase가 존재해 있다. 2. L-Tyrosine 산화효소는 액침배양한 Aspergillus oryzae의 acetone powder, cell free extract 및 배양액에도 존재하며 L-tyrosine은 ${\alpha}$-ketoglutaric acid의 첨가로 더욱 빨리 전환되었다. 3. ${\alpha}$-Ketoglutaric acid와 pyridoxal phosphate는 transamination의 amino기의 수용체로 생각되었다. 4. 이들 효소계는 L-tyrosine와 p-hydroxyphenlypyruvic acid를 homogentisic acid로 산화시켰다. 5. Ascorbic acid는 p-hydroxyphenlypyruvic가 homogentisic acid로 산화되는데 특별한 역할을 하는 것 같다. 6. L-Tyrosine-${\alpha}$-ketoglutaric acid transaminase와 p-hydroxyphenlypyruvic acid oxidase의 최적 pH는 각 각 pH $6.0{\sim}6.5$와 pH 7.5이었다.

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Assessment of vitamin $B_6$ status in Korean patients with newly diagnosed type 2 diabetes

  • Ahn, Hee-Jung;Min, Kyung-Wan;Cho, Youn-Ok
    • Nutrition Research and Practice
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    • 제5권1호
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    • pp.34-39
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    • 2011
  • The purpose of this study was to assess vitamin $B_6$ intake and status in Korean patients with newly diagnosed type 2 diabetes. Sixty-four patients with newly diagnosed type 2 diabetes and 8-11% glycated hemoglobin (A1C), along with 28 age-matched non-diabetic subjects, participated. Dietary vitamin $B_6$ intake was estimated by the 24 hour recall method and plasma pyridoxal 5'-phosphate (PLP) was measured. There was a significant difference in daily total calorie intake between the diabetic and non-diabetic groups ($1,917{\pm}376$ vs $2,093{\pm}311\;kcal$). There were no differences in intake of total vitamin $B_6$ ($2.51{\pm}0.91$ vs $2.53{\pm}0.81\;mg/d$) or vitamin $B_6$/1,000 kcal ($1.31{\pm}0.42$ vs $1.20{\pm}0.32\;mg$) between the diabetic and non-diabetic groups, and I intakes of total vitamin $B_6$ were above the Korean RDA in both groups ($180.0{\pm}57.9$ vs $179.0{\pm}65.4$). There was a higher percentage of diabetic subjects whose plasma PLP concentration was < 30 nmol/L compared to non-diabetic group. Plasma PLP levels tended to be lower in the diabetic subjects than in the non-diabetic subjects, although the difference was not statistically significant due to a large standard deviation ($80.0{\pm}61.2\;nmol/L$ vs $68.2{\pm}38.5\;nmol/L$). Nevertheless, plasma PLP levels should be monitored in pre-diabetic patients with diabetic risk factors as well as in newly diagnosed diabetic patients for long-term management of diabetes, even though this factor is not a major risk factor that contributes to the development of degenerative complications in certain patients.

3,4-Dihydroxyphenyl-L-alanine의 효소적 생산에 대한 반응첨가물의 영향

  • 이승구;노현수;홍승표;성문희
    • 한국미생물·생명공학회지
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    • 제24권2호
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    • pp.222-226
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    • 1996
  • The enzymatic synthesis of 3, 4-dihydroxyphenyl-L-alanine (L-DOPA) was examined for the effects of the reaction additives such as sodium borate, alcohol, and organic solvents. The enzyme used was tyrosine phenol-lyase of Citrobacter freundii KCTC 2006 produced in Escherichia coli. The amounts of tyrosine phenol-lyase and pyridoxal-5-phosphate were optimized to 2.0 units/ml and 0.1 mM, respectively, for the synthetic reaction. Sodium borate, a substance that forms a complex with pyrocatechol, reduced the enzyme deactivation by pyrocatechol although it seriously inhibited the enzyme activity. Among the organic solvents tested, dimethylsulfoxide, dimethylformamide, and alcohol increased the productivity of the L-DOPA synthesis. In a reaction system with 5% methanol, L-DOPA concentration increased up to 210 mM after 24 hours, and 77.1% of which was separated as precipitates. The L-DOPA was purified to 99.96% by solubilizing and recrystallyzing the precipitates.

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