156-06-9Relevant academic research and scientific papers
DIFFERENTIAL ACID-CATALYZED AROMATIZATION OF PREPHENATE, AROGENATE, AND SPIRO-AROGENATE
Zamir, Lolita O.,Tiberio, Robert,Jensen, Roy A.
, p. 2815 - 2818 (1983)
The family of closely related 2,5-cyclohexadiene, 4-hydroxy-carboxylic acid molecules are differentially labile to aromatization at mildly acidic pH in the order: prephenate > L-arogenate > spiro-arogenate.
Continuous Colorimetric Assay That Enables High-Throughput Screening of N -Acetylamino Acid Racemases
Sánchez-Carrón, Guiomar,Fleming, Toni,Holt-Tiffin, Karen E.,Campopiano, Dominic J.
, p. 3923 - 3928 (2015)
N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli l-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-d-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an l-amino acid acylase and its subsequent oxidation by an FAD-dependent l-amino acid oxidase (l-AAO). Cofactor recycling of the l-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-d-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates. (Chemical Equation Presented)
Arg305 of streptomyces l-glutamate oxidase plays a crucial role for substrate recognition
Utsumi, Tomohiro,Arima, Jiro,Sakaguchi, Chika,Tamura, Takashi,Sasaki, Chiduko,Kusakabe, Hitoshi,Sugio, Shigetoshi,Inagaki, Kenji
, p. 951 - 955 (2012)
Recently, we have solved the crystal structure of l-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward l-glutamate. By a docking simulation using l-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of l-glutamate. The activity of LGOX toward l-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various l-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate.
Characterization of aromatic aminotransferases from Ephedra sinica Stapf
Kilpatrick, Korey,Pajak, Agnieszka,Hagel, Jillian M.,Sumarah, Mark W.,Lewinsohn, Efraim,Facchini, Peter J.,Marsolais, Frédéric
, p. 1209 - 1220 (2016)
Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.
Transamination Reaction of Hydrophobic Pyridoxal with an α-Amino Acid in Functionalized Bilayer Vesicles: Co-operative Catalysis by the Imidazolyl Group and Copper(II) Ions
Murakami, Yukito,Kikuchi, Jun-ichi,Imori, Toru,Akiyoshi, Kazunari
, p. 1434 - 1436 (1984)
1-(N,N-Dihexadecylcarbamoylmethyl)-2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridinium chloride (PL+2C16) undergoes a transamination reaction with L-phenylalanine in single-walled bilayer vesicles formed from two different peptide lipids (N+C5Ala2C16 and N+C5His2C16); co-ordination of copper(II) ion to the Schiff-base intermediate results in a marked rate acceleration.
SYNTHESE DIRECTE DE PHENYLPYRUVATES CHROME TRICARBONYLE. INFLUENCES DE LA SUBSTITUTION SUR LA NATURE CETONIQUE OU ENOLIQUE DES PRODUITS ISOLES, ET DU GROUPEMENT Cr(CO)3 SUR L'EQUILIBRE TAUTOMERE.
Bihan, J. Y.-Le,Senechal-Tocquer, M.-C.,Senechal. D.,Gentric, D.,Caro, B.,et all.
, p. 3565 - 3574 (1988)
Phenylpyruvate derivatives can be readily obtained as stable enols and ketones via a new functionalization of arene Cr(CO)3 complexes.An X-Ray structure and theoretical calculations indicate that conjugation stabilizes the enol forms.The complexation of phenylpyruvic acid lowers the conjugation and shifts the keto-enolic equilibrium to the ketonic form.
Recombinant expression and characterization of a l-amino acid oxidase from the fungus Rhizoctonia solani
Hahn, Katharina,Neumeister, Katrin,Mix, Andreas,Kottke, Tilman,Gr?ger, Harald,Fischer von Mollard, Gabriele
, p. 2853 - 2864 (2017)
l-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. l-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant l-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active l-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic l-amino acids as well as methyl esters of these l-amino acids. The progress of the conversion of l-phenylalanine and l-leucine into the corresponding α-keto acids was determined by HPLC and 1H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50–100-fold by SDS treatment. Km values ranging from 0.9–10?mM and vmax values from 3 to 10?U?mg?1 were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant l-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.
Mechanistic study of the oxidation of L-phenylalanine by hexacyanoferrate(III) catalyzed by iridium(III) in aqueous alkaline medium
Goel, Anjali,Sharma, Shivani
, p. 549 - 554 (2010)
The oxidation of L-phenylalanine by hexacyanoferrate(III) (abbreviated as HCF) catalyzed by Ir(III) has been studied spectrophotometrically at 35 °C and at a constant ionic strength of 0.50 mol dm-3. The main oxidation product was identified as phenylpyruvic acid by physico-chemical and spectroscopic methods. The stoichiometry was found to be 2:1, i.e. 2 mol of hexacyanoferrate(III) reacted with 1 mol of phenylalanine. The reaction was first order with respect to both HCF and alkali concentration. The order with respect to [Phe] changed from first to zero as the concentration was increased. The effect of ionic strength was also investigated. Thermodynamic parameters were evaluated by studying the reaction at four different temperatures between 35 and 50 °C. Based on the experimental results, a suitable mechanism involving complex formation has been proposed. Springer Science+Business Media B.V. 2010.
Expression, characterization, and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum
Bloess, Svenja,Beuel, Tobias,Krüger, Tobias,Sewald, Norbert,Dierks, Thomas,Fischer von Mollard, Gabriele
, p. 2229 - 2241 (2019)
l-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate l-amino acids and produce the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here we describe the heterologous expression of LAAO4 from the fungus Hebeloma cylindrosporum without signal sequence as fusion protein with a 6His tag in Escherichia coli and its purification. 6His-hcLAAO4 could be activated by exposure to acidic pH, the detergent sodium dodecyl sulfate, or freezing. The enzyme converted 14 proteinogenic l-amino acids with l-glutamine, l-leucine, l-methionine, l-phenylalanine, l-tyrosine, and l-lysine being the best substrates. Methyl esters of these l-amino acids were also accepted. Even ethyl esters were converted but with lower activity. Km values were below 1?mM and vmax values between 19 and 39?U?mg?1 for the best substrates with the acid-activated enzyme. The information for an N-terminal aldehyde tag was added to the coding sequence. Co-expressed formylglycine-generating enzyme was used to convert a cysteine residue in the aldehyde tag to a Cα-formylglycine residue. The aldehyde tag did not change the properties of the enzyme. Purified Ald-6His-hcLAAO4 was covalently bound to a hexylamine resin via the Cα-formylglycine residue. The immobilized enzyme could be reused repeatedly to generate phenylpyruvate from l-phenylalanine with a total turnover number of 17,600 and was stable for over 40?days at 25?°C.
Specific host-guest interactions in a protein-based artificial transaminase
Haering, Dietmar,Distefano, Mark D
, p. 2461 - 2466 (2001)
Artificial enzymes can be created by covalent attachment of a catalytic active group to a protein scaffold. Recently, we assembled an artificial transaminase by conjugation of intestinal fatty acid binding protein (IFABP) with a pyridoxamine derivative via a disulfide bond; the resulting constract catalyzed a transamination reaction 200-fold faster than free pyridoxamine. To identify the origin of this increased catalytic efficiency computer modeling was first used to identify two putative residues, Y14 and R126, that were in close proximity to the γ-carboxylate group of the substrate, α-ketoglutartate. These positions were mutated to phenylalanine and methionine, respectively, and used to prepare semisynthetic transaminases by conjugation to pyridoxamine (Px) or an N-methylated derivative (Px). Kinetic analysis of the resulting constructs showed that the R126 mutation reduced substrate affinity 3- to 6-fold while the additional Y14F mutation had a negligible effect. These results are consistent with a model for substrate recognition that involves an electrostatic interaction between the cationic guanidinium group of R126 and the anionic carboxylate from the substrate. Interestingly, one of the conjugates that contains an N-methylated pyridoxamine catalyzes a transamination reaction with a kcat we have thus far obtained and is 34-fold greater than that for the free cofactor in the absence of the protein. Copyright
