• Clinical and Experimental Pediatrics
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• v.58 no.11
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• pp.407-414
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• 2015

Early-onset epileptic encephalopathies are one of the most severe early onset epilepsies that can lead to progressive psychomotor impairment. These syndromes result from identifiable primary causes, such as structural, neurodegenerative, metabolic, or genetic defects, and an increasing number of novel genetic causes continue to be uncovered. A typical diagnostic approach includes documentation of anamnesis, determination of seizure semiology, electroencephalography, and neuroimaging. If primary biochemical investigations exclude precipitating conditions, a trial with the administration of a vitaminic compound (pyridoxine, pyridoxal-5-phosphate, or folinic acid) can then be initiated regardless of presumptive seizure causes. Patients with unclear etiologies should be considered for a further workup, which should include an evaluation for inherited metabolic defects and genetic analyses. Targeted next-generation sequencing panels showed a high diagnostic yield in patients with epileptic encephalopathy. Mutations associated with the emergence of epileptic encephalopathies can be identified in a targeted fashion by sequencing the most likely candidate genes. Next-generation sequencing technologies offer hope to a large number of patients with cryptogenic encephalopathies and will eventually lead to new therapeutic strategies and more favorable long-term outcomes.

• Journal of Nutrition and Health
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• v.43 no.3
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• pp.315-323
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• 2010

The purpose of this study was to establish the selection of indicators for estimating and factors affecting the requirement of vitamin B6. There has been a need to establish the human requirements of vitamin $B_6$ since vitamin $B_6$ is thought to be involved in more than one hundred biochemical reactions as a coenzyme in the metabolism of amino acids, glucose, and lipid, and the synthesis of neurotransmitters. For the review of the literature, this study included from early findings of the sixties to studies of 2009. This study suggests that plasma pyridoxal 5' phosphate (PLP) is the best single indicator of vitamin $B_6$ status for the healthy but not for the non-healthy. Erythrocyte aspartate aminotransferase and alanine aminotransferase activation by PLP as an indirect measure and urinary 4-pyridoxic acid excretion as a direct measure are useful as supporting indicators. Bioavailability, nutrient interaction, physiological need, and chronic diseases may increase the requirement for vitamin $B_6$. However, these effects can not be quantified due to insufficient evidences.

• Journal of Community Nutrition
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• v.7 no.2
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• pp.79-84
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• 2005

A vitamin B-6 (B-6) intake higher than the current Recommended Dietary Allowance (RDA) has been found to provide an improvement in immune system. Seven premenopausal women consumed their usual diet with the exception of foods relatively high in vitamin B-6 for a total of 27 d. After 7 d, all subjects received a multivitamin supplement containing 2mg B-6 and 4 subjects were given an additional 50mg of B-6 supplement for 20 d. Lymphocyte response to phytohemagglutinin (PHA) was measured before and after the supplementation. To determine the effect of different forms of B-6 on lymphocyte proliferation, cell culture media supplemented with pyridoxal (PL) and PLP, as well as B-6 free media, were tested. A 50mg B-6 supplement significantly increased vitamin B-6 status. There was no further enhancement on lymphocyte proliferation when subjects were taking an additional 50mg of vitamin B-6 supplement. In general, lymphocyte proliferation in media with either PLP or PL did not show any prominent difference. These [m-dings suggest that there may be no further benefits of a B-6 dose beyond twice that of the current RDA on lymphocyte proliferation. Further studies are necessary to examine the effect of the B-6 intake level on activities of enzymes involved in cellular B-6 metabolism in lymphocytes to provide substantial insight into the mechanisms underlying the role of B-6 in the lymphocyte proliferation.

• Microbiology and Biotechnology Letters
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• v.47 no.1
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• pp.139-147
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• 2019

Glutamate decarboxylase catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), contributing to pH homeostasis through proton consumption. The reaction is the first step toward the GABA shunt. To date, the enzymes involved in the glutamate metabolism of Photobacterium damselae subsp. piscicida have not been elucidated. In this study, an open reading frame of P. damselae subsp. piscicida, showing homology to the glutamate decarboxylase or putative pyridoxal-dependent aspartate 1-decarboxylase genes, was isolated and cloned into an expression vector to produce the recombinant enzyme. Preliminary gas chromatography-mass spectrometry characterization of the purified recombinant enzyme revealed that it catalyzed not only the decarboxylation of glutamate but also the transamination of GABA. This enzyme of P. damselae subsp. piscicida could be bifunctional, combining decarboxylase and transaminase activities in a single polypeptide chain.

• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.9-17
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• 2021

A γ-aminobutyric acid (GABA)-producing microorganism was isolated from galchi (hairtail fish, Trichiurus lepturus) jeotgal, a Korean salted and fermented seafood. The G144 isolate produced GABA excessively when incubated in MRS broth containing monosodium glutamate (MSG, 3%, w/v). G144 was identified as Lactobacillus brevis through 16S rRNA and recA gene sequencing. gadB and gadC encoding glutamate decarboxylase (GAD) and glutamate/GABA antiporter, respectively, were cloned and gadB was located downstream of gadC. The operon structure of gadCB was confirmed by reverse transcription (RT)-polymerase chain reaction. gadB was overexpressed in Escherichia coli and recombinant GAD was purified and its size was 54.4 kDa as evidenced by SDS-PAGE results. Maximum GAD activity was observed at pH 5.0 and 40℃ and the activity was dependent on pyridoxal 5'-phophate. The K_m and V_max of GAD were 8.6 mM and 0.01 mM/min, respectively.

• Parasites, Hosts and Diseases
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• v.21 no.1
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• pp.49-57
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• 1983

The distribution and Properties of branched chain amino acid aminotransferase (EC 2.6. 1.42) was investigated in adult Fasciola hepatica. Fascicla hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of branched chain amino acid aminotransferase was measured by the method of Ichihara and Koyama (1966) . Isozyme patterns of this enzyule was also examined by DEAE-cellulose column chromatography. The results obtained were as follows; 1. The activity in homogenate was found to be 12.69 units/g wet tissue. The activity of this enzyme was relatively high compared with those in rat tissues. 2. The distribution of branched chain amino acid aminotransferase in the subcellular organelles showed that 87.8% of the activity was in cytosolic, 10.9% in mitochondrial and 1.3% was in nuclear fraction. 3. Cytosolic fraction of Fasciola hepatica contained Enzyme I, but not Enzyme II and III, of branched chain amino acid aminotransferase. Ensyme I was eluted by 50mM phosphate buffier from DEAE-cellulose column and catalyzed the transamination of all three branched chain amino acids. 4. The Enzyme I was purified about 22-folds increase in specific activity after chromatography on DEAE-cellulose. 5. The best substrate among three amino acids (leucine, isoleucine and valise) was L-isoleucine. 6. The optimal temperature of Enzyme I was $45^{\circ}C$ and the optimal pH was 8.2. 7. The Km value for leucine of Enzyme I was 4.17 mM. 8. The Km values for a-ketoglutarate and pyridoxal phosphate of Enzyme I were 0.41mM and $4.76{\times}10^{-3}{\;}mM$, respectively.

• Journal of Applied Biological Chemistry
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• v.53 no.3
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• pp.132-138
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• 2010

A gene encoding a putative alanine racemase in B. pseudomycoides was cloned and expressed in Escherichia coli BL21(DE3) using a pET-21 vector harbouring 6xHistidine tag. Affinity purification of the recombinant alanine racemase with a nickel resin resulted in one band by SDS-PAGE analysis. The purified enzyme showed a molecular weight of 46 kDa. The enzyme was the most active toward L-alanine and secondly D-alanine, implying that the enzyme is an alanine racemase. D-cysteine significantly inhibited the enzyme activity and also L-cysteine to a lesser extent. The enzyme was considerably activated by addition of pyridoxal-5'-phosphate (PLP), showing that 73% increase in activity was observed at 0.3 mM, compared to control. The enzyme was the most active at pH 9.0 and more stable at alkaline pHs than acidic pHs.

• BMB Reports
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• v.32 no.5
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• pp.480-485
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• 1999

Tyrosine phenol-lyase of thermophilic Symbiobacterium sp. SC-1, which is obligately and symbiotically dependent on thermophilic Bacillus sp. SK-1, was purified and characterized. The enzyme is composed of four identical subunits and contains approximately 1 mol of pyridoxal 5'-phosphate (PLP) per mol subunit as a cofactor. The enzyme showed absorption maxima at 330 and 420 nm, and lost this absorption profile by treatment with phenylhydrazine. The apparent dissociation constsnt, $K'_D$, for PLP was determined with the apoenzyme to be about $1.2\;{\mu}M$. The isoelectric point was 4.9. The optimal temperature and pH for the $\alpha,\beta$-elimination of L-tyrosine were found to be $80^{\circ}C$ and pH 8.0, respectively. The substrate specificity of the enzyme was very broad: L-amino acids including L-tyrosine, 3,4-dihydroxyphenyl-L-alanine (L-DOPA), L-cysteine, L-serine, S-methyl-L-cysteine, $\beta$-chloro-L-alanine, and S-(o-nitrophenyl)-L-cysteine all served as substrates. D-Tyrosine and D-serine were also decomposed into pyruvic acid and ammonia at rates of 7% and 31% relative to their corresponding L-enantiomers, respectively. D-Alanine, which was inert as a substrate in a, $\beta$-elimination, was the only D-amino acid racemized by the enzyme. The $K_m$ values for L-tyrosine, L-DOPA, S-(o-nitrophenyl)-L-cysteine, $\beta$-chloro-L-alanine, and S-methyl-L-cysteine were 0.19, 9.9, 0.36, 12, and 5.5 mM, respectively.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.505.2-506
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• 1986

L-Azetidine-2-carboxylic acid (A-2-C), a four-membered cyclic imino acid has been identified in certain plants, and the microorganism Actinoplanes ferrugineus. The imino acid A-2-C has a physiological significance as an antgaonist of proline during peptide synthesis. The biosynthetic mechanism for the formation of A-2-C has not been studied in any detail. By using various amino acids such as methionine and S-adenosyl-L-methionine labeled with deuterium or carbon-14, the details of the biosynthetic pathway and a possible mechanism for the formation of L-A-2-C in .4. ferrugineus have been unravelled, Both in vivo and in vitro experimental results suggest the biosynthesis of L-A-2-C is mediated by a confactor containing a carbonyl group, probably pyridoxal Phosphate. S-Adenosyl-L-methionine, which seems to be the direct biosynthetic substrate, has undergone a f-displacement by an ${\alpha}$-amino group of the amino acid portion of the substrate S-adenosyl-L-methionine potentially via a vinylglycine intermediate. The overall stereochemical events at the ${\beta}$-carbon of the substrate have been shown to inversion of configuration. The overall stereochemical events at the -position of the sub- strate have also been shown to occur with inversion of configuration. The ${\beta}$, ${\gamma}$-elimination reaction of the substrate seems to follow a cisoidal-type mechanism and the addition portion of the reaction a transoidal-type mechanism . The assignment of the proton NMR of A-2-C has been deduced by apply- ing NOE difference experiments, Gd(III) line-broadening experiments and 2D-NOESY experiments of regio-and stereospecificially deuterated A-2-C's.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1586-1592
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• 2016

Klebsiella pneumoniae is a gram-negative, non-motile, rod-shaped, and encapsulated bacterium in the normal flora of the intestines, mouth, skin, and food, and has decarboxylation activity, which results in generation of diamines (cadaverine, agmatine, and putrescine). However, there is no specific information on the exact mechanism of decarboxylation in K. pnuemoniae. Specifically lysine decarboxylases that generate cadaverine with a wide range of applications has not been shown. Therefore, we performed a functional study of lysine decarboxylases. Enzymatic characteristics such as optimal pH, temperature, and substrates were examined by overexpressing and purifying CadA and LdcC. CadA and LdcC from K. pneumoniae had a preference for L-lysine, and an optimal reaction temperature of 37℃ and an optimal pH of 7. Although the activity of purified CadA from K. pneumoniae was lower than that of CadA from E. coli, the activity of K. pneumoniae CadA in whole cell bioconversion was comparable to that of E. coli CadA, resulting in 90% lysine conversion to cadaverine with pyridoxal 5'-phosphate L-lysine.