• BMB Reports
• /
• 제33권1호
• /
• pp.82-88
• /
• 2000

A homologous gene to alanine racemase was cloned from a hyperthermophilic bacterium, Aquifex pyrophilus. The cloned gene encodes a protein of 341 amino acids, which has a significant homology to alanine racemase of Bacillus stearothermophilus, Lactobacillus brevis, and E. coli. When the gene was expressed in Escherichia coli, it produced a 40 kDa protein. The purified protein contains one mole pyridoxal 5-phosphate per one mole of protein, which is essential for catalytic activity of alanine racemase. The purified protein catalyzed racemization of L-alanine to D-alanine, or vice versa, indicating that the cloned gene encoded alanine racemase. It also showed significant racemization activity against L-serine and ${\alpha}-aminobutylic$ acid. The A. pyrophilus alanine racemase showed strong thermostability, and it maintained catalytic activity in the presence of organic solvents.

• BMB Reports
• /
• 제33권1호
• /
• pp.43-48
• /
• 2000

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It converts the neurotransmitter 4-aminobutyric acid to succinic semialdehyde. In order to study the structural and functional aspects of catalytically active Cys residues of pig brain 4-aminobutyrate aminotransferase, we purified the active form in E. coli by coproduction of thioredoxin. The structural arrangement for functional requirements of a dimeric protein using a bifunctional sultbydryl reagent was then characterized, and the spatial proximity between the essential SH groups and a cofactor (pyridoxal-5'-phosphate) binding site was determined. The bifunctional sultbydryl reagent DMDS reacted with the enzyme at the ratio of one molecule per enzyme dimer. This resulted in an approximately 50% loss of enzymatic activity. The spatial proximity of the distance between the essential SH groups and the cofactor-binding site was determined by the energy transfer measurement technique. The result (approximate 20 ${\AA}$) suggested that cross-linking of two sulfhydryl groups with DMDS is not near a PLP binding site.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1995년도 춘계학술대회
• /
• pp.73-73
• /
• 1995

We investigated the effect of derivatized phospholipid, P-pyridoxyl dipalmiuylphosphatidylethanolamine (P-pyr-DPPE), on the catalytic activity of purified porcine brain glutamate decarboxylase(GAD) which catalyzes the synthesis of GABA known as major inhibitory neurotransmitter in CNS. When the P-pyr-DPPE was incorporated into dipalmitdylphosphatidylcholine(DPPC) or phosphatidylserine(PS) vesicles, these vesicles enhanced the catalytic activity of GAD. P-pyr-DPPE also interacted with apoglutamate decarboxylase(apoGAD) and produced the free pyridoxal-5-phosphate(PLP) which is the natural cofactor of GAD. This result indicated that apoGAD catalyzed the cleavage reaction of the P-pyridoxyl moiety of the derivatized phopholipid to generate free PLP, and then free PLP bound to the apoGAD resulting in restroration of the catalytic activity of the enzyme.

• Journal of Nutrition and Health
• /
• 제33권3호
• /
• pp.263-270
• /
• 2000

The purpose of this study was to evaluate the concentration of vitamin B6 in 16 pregnant-infant pairs and 15 nonpregnant women and to investigate the relationships between vitamin B6 status of maternal-umbilical cord plasma and pregnancy outcomes. dietary intake was obtained from semiquantitative frequency questionnaire. The daily mean energy and protein intakes were higher than the recommended dietary allowance, while daily vitamin B6 was only 74% of RDA in pregnant and 73% of RDA in nonpregnant women. The main sources of vitamin B6 were vegetables and fruits in pregnant women, while cereal and starch in nonpregnant women. The plasma PLP and PL levels of pregnant women were 14.85nmol/l and 20.56nmol/l, significantly lower than those of nonpregnant women. the PLP/PL ratios of pregnant and nonpregnant women were 1.65 and 0.33, indicating that the levels of vitamin B6 was altered during pregnancy. The PLP and PL levels of umbilical cord plasma were 63.55nmol/l and 32.25nmol/l, respectively. The vitamin B6 levels of umbilical cord plasma were significantly higher than that of maternal plasm. This finding indicates that the uptake of vitamin B6 in the fetus may be due to an active placental transport mechanism. The PLP level of maternal plasma correlated positively with that of umbilical cord plasma, showing the PLP concentration of umbilical cord plasma is affected by maternal vitamin B6 status. The maternal plasma PL level showed a positive correlation to infant birth weight. The positive association has bee also found between plasma PL level of umbilical cord and Apgar 1 min score.

• Journal of Microbiology and Biotechnology
• /
• 제16권9호
• /
• pp.1434-1440
• /
• 2006

The stable anchorage of pyridoxal 5'-phosphate (PLP) in the active site of D-amino acid transaminase (D-AT) is crucial for the enzyme catalysis. The three-dimensional structure of D-AT revealed that Glu32 is one of the active site groups that may playa role in PLP binding. To prove the role of Glu32 in PLP stability, we firstly checked the rate of the potential rate-limiting step. The kinetic analysis showed that the rate of the ${\alpha}$-deprotonation step reduced to 26-folds in E32A mutant enzyme. Spectral analyses of the reaction of D-AT with D-serine revealed that the E32A mutant enzyme failed to stabilize the key enzyme-substrate intermediate, namely a quinonoid intermediate (E-Q). Finally, analysis of circular dichroism (CD) on the wild-type and E32A mutant enzymes showed that the optical activity of PLP in the enzyme active site was lost by the removal of the carboxylic group, proving that Glu32 is indeed involved in the cofactor anchorage. The results suggested that the electrostatic interaction network through the groups from PLP, Glu32, His47, and Arg50, which was observed from the three-dimensional structure of the enzyme, plays a crucial role in the stable anchorage of the cofactor to give necessary torsion to the plane of the cofactor-substrate complex.

• BMB Reports
• /
• 제38권6호
• /
• pp.703-708
• /
• 2005

We cloned and expressed human pyridoxal-5'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin $B_6$, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin $B_6$ abnormalities.

• 한국식품영양학회지
• /
• 제23권2호
• /
• pp.171-177
• /
• 2010

Serratia marcescens catabolic threonine dehydratase는 streptomycin sulfate treatment, Sephadex G-200 gel filtration, AMP-Sepharose 4B affinity chromatography 등의 방법으로 정제하였는데, 최종 단계에서 회수율은 15.5%이었으며 50배 정제되었다. Native 분자량은 native pore gradient polyacrylamide gel electrophoresis(PAGE) 방법으로는 120,000이었다. SDS-PAGE에 의한 subunit의 분자량은 30,000이었고, 즉 S. marcescens 효소는 4개의 동일한 subunit으로 구성된 homo-tetrameric protein임이 판명되었다. S. marcescens 효소의 L-threonine에 대한 Km값은 AMP가 있는 조건에서 7.3 mM, AMP가 없는 조건에서 92 mM이었다. S. marcescens 효소는 효소 1 mole 당 각각 2 mole의 pyridoxal 5'-phosphate(PLP), 16개의 free-SH group을 가지고 있었다. S. marcescens 효소는 AMP의 존재 하에서 $\alpha$-ketobutyrate, pyruvate, glyoxylate, phosphoenol pyruvate(PEP)에 의해 효소 활성이 억제되었으며, cAMP와 ADP에 의해서는 효소 활성이 증가되었다. 효소학적 성질면에서 S. marcescens 효소는 E. coli 효소보다는 S. typhimurium 효소와 유사하였다. 한편, E. coli 효소는 cAMP에 의하여 효소 활성이 증가되고, S. typhimurium 효소는 ADP에 의해 효소 활성이 증가되는 것과 다르게, S. marcescens 효소는 cAMP와 ADP 모두 효소 활성이 증가되었다. 따라서 이상의 연구 결과들은 세 enteric bacteria의 catabolic threonine dehydratase가 서로 작은 차이점이 있다는 것을 반영하며, 이러한 사실을 규명하기 위해서는 향후 보다 심층적인 연구를 수행하여야 할 것으로 사료된다.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 1986년도 추계학술대회
• /
• pp.515.1-515
• /
• 1986

Thermostable tryptophanase was extracted from a thermophilie bacterium, strain T which was absolutely symbiotic with strain 5. The enzyme was purified 14.7 fold with 5.8% yield by chromatographies using ion exchange, gel filtration, and hydrophobic interaction columns, followed by high performance liquid chromatography on hydroxyapatite column. The purified enzyme has a molecular weight of approximately 210,000 estimated by gel filtration column chromatography, and the molecular weight of subunit was determined by SDS polyacrylamide gel electrophoresis to be 46,000, which indicates that the native enzyme is made of four homologous subunits. The tryptophanase was stable at 65o0 and the optimum temperature for the enzyme activity for 20 min reaction was 70$^{\circ}C$. The purified enzyme activity for 20 min ieaction was 70$^{\circ}C$. The purified enzyme catalyzed the degradation of L-tryptophan into indole, pyruvate and ammonia in the presence of pyridoxal phosphate. 5-Hydroxy-Ltryptophan, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-L-cysteine, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-Lcysteine, and L-serine were also used as substrates to form pyruvate. The amino acid composition of the tryptophanase was determined, and found to contain a high percentage of hydrophobic amino acids, especially in the proline content, which was much higher than that of Escherichia coli tryptophanase. In addition, the 35N-terminal amino acid sequence of the tryptophanase was completely different from that of E. coli tryptophanase.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권3호
• /
• pp.180-185
• /
• 2005

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase from chromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1mM NBS, chloramine-T, $\rho-CMB,\;\rho-HMB$ and iodine, and was strongly inhibited by 1mM PMSF and pyridoxal 5'-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by $\rho-CMB$ was also reversed by 1mM cysteine-HCI, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase from C violaceum YK 391 was assumed to be a thiol enzyme.

• Journal of Microbiology and Biotechnology
• /
• 제17권9호
• /
• pp.1579-1584
• /
• 2007

5-Aminolevulinate (ALA) synthase (E.C. 2.3.1.37), which mediates the pyridoxal phosphate-dependent condensation of glycine and succinyl-CoA, encoded by the Rhodobacter sphaeroides hemA gene, enables Escherichia coli strains to produce ALA at a low level. To study the effect of the enhanced C4 metabolism of E. coli on ALA biosynthesis, NADP-dependent malic enzyme (maeB, E.C. 1.1.1.40) was coexpressed with ALA synthase in E. coli. The concentration of ALA was two times greater in cells coexpressing maeB and hemA than in cells expressing hemA alone under anaerobic conditions with medium containing glucose and glycine. Enhanced ALA synthase activity via coupled expression of hemA and maeB may lead to metabolic engineering of E. coli capable of large-scale ALA production.