- Production of pyridoxal phosphate by a mutant strain of Schizosaccharomyces pombe.
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Conditions for extracellular production of vitamin B6 compounds (B6), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leul strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B6 and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B6 and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H202 increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and, in contrast, increased pyridoxine.
- Chumnantana,Hirose,Baba,Yagi
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- Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase
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We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[2H 8] Phenylalanine was converted to [2H8] phenylacetaldehyde and [2H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5′-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.
- Sakai, Miwa,Hirata, Hiroshi,Sayama, Hironori,Sekiguchi, Kazuya,Itano, Hiroaki,Asai, Tatsuo,Dohra, Hideo,Hara, Masakazu,Watanabe, Naoharu
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- Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase
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The mechanisms of pyridoxal 5′-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine Cα-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of Cα-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the Cα-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C α-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a Cα-H pKa that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.
- Hill, Melissa P.,Carroll, Elizabeth C.,Vang, Mai C.,Addington, Trevor A.,Toney, Michael D.,Larsen, Delmar S.
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supporting information; experimental part
p. 16953 - 16961
(2011/02/16)
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- Chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids
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The mechanism of chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids is studied by kinetic measurements. The Schiff bases are shown to be fairly stable in neutral media. In acid media, the Schiff bases a
- Pishchugin,Tuleberdiev
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p. 1225 - 1229
(2008/12/22)
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- Metal ion inhibition of nonenzymatic pyridoxal phosphate catalyzed decarboxylation and transamination
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Nonenzymatic pyridoxal phosphate (PLP) catalyzed decarboxylations and transaminations have been revisited experimentally. Metal ions are known to catalyze a variety of PLP-dependent reactions in solution, including transamination. It is demonstrated here that the rate accelerations previously observed are due solely to enhancement of Schiff base formation under subsaturating conditions. A variety of metal ions were tested for their effects on the reactivity of the 2-methyl-2-aminomalonate Schiff bases. All were found to have either no effect or a small inhibitory one. The effects of Al3+ were studied in detail with the Schiff bases of 2-methyl-2-aminomalonate, 2-aminoisobutyrate, alanine, and ethylamine. The decarboxylation of 2-methyl-2-aminomalonate is unaffected by metalation with Al3+, while the decarboxylation of 2-aminoisobutyrate is inhibited 125-fold. The transamination reaction of ethylamine is 75-fold slower than that of alanine. Ethylamine transamination is inhibited 4-fold by Al3+ metalation, while alanine transamination is inhibited only 1.3-fold. Metal ion inhibition of Schiff base reactivity suggests a simple explanation for the lack of known PLP dependent enzymes that make direct mechanistic use of metal ions. A comparison of enzyme catalyzed, PLP catalyzed, and uncatalyzed reactions shows that PLP dependent decarboxylases are among the best known biological rate enhancers: decarboxylation occurs 1018-fold faster on the enzyme surface than it does free in solution. PLP itself provides the lion's share of the catalytic efficiency of the holoenzyme: at pH 8, free PLP catalyzes 2-aminoisobutyrate decarboxylation by ～1010-fold, with the enzyme contributing an additional ～108-fold.
- Zabinski,Toney
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p. 193 - 198
(2007/10/03)
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- Catalytic Efficiency of Functionalized Vesicles in the Transamination of Pyridoxal-5'-phosphate with a Hydrophobic Amino Acid
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The transamination reaction of pyridoxal-5'-phosphate (PLP) with N-dodecyl-L-alaninamide (AlaC12) was investigated in an aqueous phosphate-borate buffer at pH 7.0, μ 0.10 (KCl), and 30.0+/-0.1 deg C in the presence of single-walled vesicles of N,N-ditetradecyl-Nα-(6-trimethylammoniohexanoyl)-L-histidinamide bromide (N+C5His2C14).The electrostatic and hydrophobic interactions between the vesicles and the reactants resulted in incorporation of PLP and AlaC12 into polar and hydrophobic domains of the vesicles, respectively, in the Schiff-base formation process.The isomerization of the aldimine Schiff-base to the correspeonding ketimine Schiff-base was confirmed to be the rate-determining step in the transamination process.The reaction site in the vesicular system was found to be equivalent in polarity to dioxane-water (7:3 v/v).However, the overall reaction rate in the vesicles was enhanced 230-fold relative to that in dioxane-water (7:3 v/v).A hydrophobic and suitably polar microenvironment constructed at the reaction site is responsible for such a marked rate-enhancement.In addition, each vesicle of N+C5His2C14 provided functional (imidazolyl) groups in its hydrogen-belt domain to catalyze the intramolecular prototropic shift to yield the ketimine Schiff-base.The microenvironmental effects of molecular assemblies of N,N-ditetradecyl-Nα-(6-trimethylammoniohexanoyl)-L-alaninamide bromide and CTAB on the overall transamination were also discussed.
- Murakami, Yukito,Nakano, Akio,Akiyoshi, Kazunari
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p. 3004 - 3012
(2007/10/02)
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