• BMB Reports
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• v.38 no.6
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• pp.703-708
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• 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.

• Molecules and Cells
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• v.41 no.12
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• pp.1033-1044
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• 2018

As sessile organisms, plants have evolved to adjust their growth and development to environmental changes. It has been well documented that the crosstalk between different plant hormones plays important roles in the coordination of growth and development of the plant. Here, we describe a novel recessive mutant, mildly insensitive to ethylene (mine), which displayed insensitivity to the ethylene precursor, ACC (1-aminocyclopropane-1-carboxylic acid), in the root under the dark-grown conditions. By contrast, mine roots exhibited a normal growth response to exogenous IAA (indole-3-acetic acid). Thus, it appears that the growth responses of mine to ACC and IAA resemble those of weak ethylene insensitive (wei) mutants. To understand the molecular events underlying the crosstalk between ethylene and auxin in the root, we identified the MINE locus and found that the MINE gene encodes the pyridoxine 5′-phosphate (PNP)/pyridoxamine 5′-phosphate (PMP) oxidase, PDX3. Our results revealed that MINE/PDX3 likely plays a role in the conversion of the auxin precursor tryptophan to indole-3-pyruvic acid in the auxin biosynthesis pathway, in which TAA1 (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1) and its related genes (TRYPTOPHAN AMINOTRANSFERASE RELATED 1 and 2; TAR1 and TAR2) are involved. Considering that TAA1 and TARs belong to a subgroup of PLP (pyridoxal-5′-phosphate)-dependent enzymes, we propose that PLP produced by MINE/PDX3 acts as a cofactor in TAA1/TAR-dependent auxin biosynthesis induced by ethylene, which in turn influences the crosstalk between ethylene and auxin in the Arabidopsis root.

• Journal of Life Science
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• v.18 no.4
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• pp.585-589
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• 2008

Chronophin is a phosphatase responsible for the dephosphorylation of cofilin, which regulates the rearrangement of actin cytoskeleton. It is also known as a phosphatase for pyrodoxal 5'-phosphate (PLP), an active form of vitamin $B_6$, and maintains the level of PLP in the cytoplasm. Since this phosphatase belongs to a HAD subfamily containing a cap domain, it is expected to undergo a conformational change for the binding of a substrate. However, the crystal structure of chronophin has a disulfide bridge between the cap and core domains preventing a movement of the cap domain against the core domain. It is possible that the disulfide bond between C91 and C221 was formed by an oxidation during the crystallization. Here, we obtained chronophin crystals under a reduced condition and determined the crystal structure. This reduced chronophin does not contain a disulfide bridge and shows a closed conformation like the oxidized form. It implies that an active chronophin binds its substrate under the closed conformation without the disulfide bond and shows a high substrate specificity in the cell.

• 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.

• BMB Reports
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• v.33 no.1
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• pp.43-48
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• 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.

• Journal of Nutrition and Health
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• v.35 no.3
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• pp.322-331
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• 2002

This study was carried out to evaluate the effect of vitamin $B_{6}$ intake by normal term delivery pregnant women on the concentration of vitamin $B_{6}$ in the material plasma, the umbilical cord plasma, and the placental tissue. Dietary intake data were obtained from a semi-quantitative frequency questionnaire. The daily mean energy and protein intakes were 2189.5 kcal (93.2% of RDA) and 79.3 g (113.3% of RDA), respectively. The average daily vitamin $B_{6}$ intake was 1.7 mg (91.4% of RDA) for the pregnant women. Their main sources of vitamin $B_{6}$ were cereal & starch (50%), and vegetables & fruits (33%). The pyridoxal phosphate (PLP) concentration of the maternal plasma, the umbilical cord plasma, and the placenta were 16.7 $\pm$ 4.1 nmol/1, 61.3 $\pm$ 19.8 nmol/l and 898.6 $\pm$ 159.2 ng/g, respectively. The PLP level was the highest in the placenta. The PLP level of the maternal plasma was significantly lower than the of the umbilical cord plasma (p < 0.001). The PLP level of maternal plasma correlated positively with that of the placenta (p < 0.0001) and the umbilical cord plasma (p < 0.05). Also the PLP level of the placenta correlated positively with that of the umbilical cord plasma (p < 0.05). These findings indicate that the vitamin $B_{6}$ nutritional status of the fetus is affected by placental vitamin $B_{6}$ levels, and that the placental vitamin B$_{6}$ levels reflect the maternal vitamin $B_{6}$ status. The umbilical cord plasma PLP level showed a positive correlation with the gestational length (p < 0.05). A negative association was observed between the PLP level showed of the umbilical cord plasma and the pregnancy weight gain (p < 0.03). The results suggest that the transfer of PLP from maternal plasma to the placental tissue could be an active transport, white the transfer of PLP from the placenta to the fetus is by means of simple diffusion. Thus, neonatal vitamin $B_{6}$ nutrition is influenced by the maternal nutritional status.

• Journal of Life Science
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• v.17 no.7 s.87
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• pp.970-975
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• 2007

For the screening of ${\gamma}-aminobutyric$ acid (CABA)-producing bacteria, 86 bacterial strains which produce GABA were isolated from Kimchi and Salted fisk .Among these, three strains designated AML15, AML45-1, AML72 with relatively high GABA productivity were selecled by thin layer chromatography (TLC). To elucidate the relationship between isolated strains and the genus Lactobacillus, their 16S rDNA sequence were examined. The result of their DNA sequences showed 99% similarity with Lactobacillus brevis ATCC 367. On the basis of the these results, isolated strains were identified as Lactobacillus brevis and designated L. brevis AML15. In order to determine the optimum conditions for GABA production, the isolated strains were cultivated in pyridoxal phosphate (PLP) and monosodium glutami. acid (MSG). Results showed that L. brevis AML15 had the highest CABA productivity with 10,424 $nM/{\mu}l$ concentration in MRS broth containing 5% (w/v) MSG and 10 ${\mu}M$ PLP at pH 5.0. The results imply that L. brevis AML15 has the potential to be developed as a strain for GABA hyper-production.

• The Korean Journal of Food And Nutrition
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• v.23 no.2
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• pp.171-177
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• 2010

The catabolic threonine dehydratase from Serratia marcescens ATCC 25419 was purified to homogeniety using Sephadex G-200 gel filtration and AMP-Sepharose 4B affinity chromatography. The molecular weight of the native enzyme was 120,000 by native pore gradient PAGE. The enzyme was composed of four identical subunits with subunit molecular weights of 30,000 by SDS-PAGE. The Km values of the enzyme for L-threonine with and without AMP were 7.3 and 92 mM, respectively. There were 2 moles of pyridoxal phosphate and 16 moles of free -SH groups per 1 mole of enzyme. The enzyme was inhibited by $\alpha$-ketobutyrate, pyruvate, glyoxylate, and phosphoenol pyruvate(PEP) in the presence of AMP, yet stimulated by cAMP and ADP. For enzyme properties in comparison with S. marcescens, E. coli, and S. typhimurium enzyme, such as the PLP content, number of free sulfhydryl groups, and existence of ADP binding site, the S. marcescens enzyme was more similar to the S. typhimurium enzyme than the E. coli enzyme. Of the three enteric bacteria, the E. coli and S. typhimurium enzyme was increased the activity by ADP and cAMP, respectively, but only the S. marcescens enzyme was increased the activity by both ADP and cAMP. Therefore, the subtle differences in the properties between enzymes from the three enteric bacteria may represent minor structural differences among these enzymes and warrants further study.

• 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.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.55-58
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• 2008

Alanine racemase, a bacterial enzyme belonging to the fold-type III group of pyridoxal 5'-phosphate (PLP)-dependent enzymes, has been shown to catalyze the interconversion between L- and D-alanine. The alanine racemase from the pathogenic bacterium Enterococcus faecalis v583 has been overexpressed in E. coli and was shown to crystallize an enzyme at 295 K, using polyethylene glycol (PEG) 8000 as a precipitant. X-ray diffraction data to $2.5{\AA}$ has been collected using synchrotron radiation. The crystal is a member of the orthorhombic space group, $C222_1$ with unit cell parameter of a=94.634, b=156.516, $c=147.878{\AA},\;and\;{\alpha}={\beta}={\gamma}=90{\AA}$. Two or three monomers are likely to be present in the asymmetric unit, with a corresponding $V_m\; of\;3.38{\AA}^3\;Da^{-1}\;and\;2.26{\AA}^3\;Da^{-1}$ and a solvent content of 63.7% and 45.5%, respectively.