- Coupling between D-3-phosphoglycerate dehydrogenase and D-2-hydroxyglutarate dehydrogenase drives bacterial L-serine synthesis
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L-Serine biosynthesis, a crucial metabolic process in most domains of life, is initiated by D-3-phosphoglycerate (D-3-PG) dehydrogenation, a thermodynamically unfavorable reaction catalyzed by D-3-PG dehydrogenase (SerA). D-2-Hydroxyglutarate (D-2-HG) is traditionally viewed as an abnormal metabolite associated with cancer and neurometabolic disorders. Here, we reveal that bacterial anabolism and catabolism of D-2-HG are involved in L-serine biosynthesis in Pseudomonas stutzeri A1501 and Pseudomonas aeruginosa PAO1. SerA catalyzes the stereospecific reduction of 2-ketoglutarate (2-KG) to D-2-HG, responsible for the major production of D-2-HG in vivo. SerA combines the energetically favorable reaction of D-2-HG production to overcome the thermodynamic barrier of D-3-PG dehydrogenation. We identified a bacterial D-2-HG dehydrogenase (D2HGDH), a flavin adenine dinucleotide (FAD)-dependent enzyme, that converts D-2-HG back to 2-KG. Electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) are also essential in D-2-HG metabolism through their capacity to transfer electrons from D2HGDH. Furthermore, while the mutant with D2HGDH deletion displayed decreased growth, the defect was rescued by adding L-serine, suggesting that the D2HGDH is functionally tied to L-serine synthesis. Substantial flux flows through D-2-HG, being produced by SerA and removed by D2HGDH, ETF, and ETFQO, maintaining D-2-HG homeostasis. Overall, our results uncover that D-2-HG–mediated coupling between SerA and D2HGDH drives bacterial L-serine synthesis.
- Zhang, Wen,Zhang, Manman,Gao, Chao,Zhang, Yipeng,Ge, Yongsheng,Guo, Shiting,Guo, Xiaoting,Zhou, Zikang,Liu, Qiuyuan,Zhang, Yingxin,Ma, Cuiqing,Tao, Fei,Xu, Ping
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Read Online
- Integrating error-prone PCR and DNA shuffling as an effective molecular evolution strategy for the production of α-ketoglutaric acid by l-amino acid deaminase
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l-Amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acids from l-amino acids. In a previous study, a whole cell biocatalyst with the l-amino acid deaminase (pm1) from Proteus mirabilis was developed for the one-step production of α-ketoglutarate (α-KG) from l-glutamic acid, and the α-KG titer reached 12.79 g L-1 in a 3 L batch bioreactor. However, the product α-KG strongly inhibited pm1 activity, and the titer of α-KG was comparatively lower than expected. Therefore, in this study, multiple rounds of error-prone polymerase chain reaction (PCR) and gene shuffling were integrated for the molecular engineering of pm1 to further improve the catalytic performance and α-KG titer. A variant (pm1338g4), which contained mutations in 34 amino acid residues, was found to have enhanced catalytic efficiency. In a batch system, the α-KG titer reached 53.74 g L-1 when 100 g of monosodium glutamate was used as a substrate. Additionally, in a fed-batch biotransformation system, the maximum α-KG titer reached 89.11 g L-1 when monosodium glutamate was continuously fed at a constant rate of 6 g L-1 h-1 (from 4 to 23 h) with an initial concentration of 50 g L-1. Analysis of the kinetics of the mutant variant showed that these improvements were achieved due to enhancement of the reaction velocity (from 56.7 μM min-1 to 241.8 μM min-1) and substrate affinity (the Km for glutamate decreased from 23.58 to 6.56 mM). A possible mechanism for the enhanced substrate affinity was also evaluated by structural modeling of the mutant. Our findings showed that the integration of error-prone PCR and gene shuffling was an effective method for improvement of the catalytic performance of industrial enzymes.
- Hossain, Gazi Sakir,Shin, Hyun-Dong,Li, Jianghua,Wang, Miao,Du, Guocheng,Liu, Long,Chen, Jian
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Read Online
- A new l-arginine oxidase engineered from l-glutamate oxidase
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The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.
- Yano, Yoshika,Matsuo, Shinsaku,Ito, Nanako,Tamura, Takashi,Kusakabe, Hitoshi,Inagaki, Kenji,Imada, Katsumi
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p. 1044 - 1055
(2021/04/14)
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- From a dimer to a monomer: Construction of a chimeric monomeric isocitrate dehydrogenase
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Many isocitrate dehydrogenases (IDHs) are dimeric enzymes whose catalytic sites are located at the intersubunit interface, whereas monomeric IDHs form catalytic sites with single polypeptide chains. It was proposed that monomeric IDHs were evolved from dimeric ones by partial gene duplication and fusion, but the evolutionary process had not been reproduced in laboratory. To construct a chimeric monomeric IDH from homo-dimeric one, it is necessary to reconstitute an active center by a duplicated region; to properly link the duplicated region to the rest part; and to optimize the newly formed protein surface. In this study, a chimeric monomeric IDH was successfully constructed by using homo-dimeric Escherichia coli IDH as a start point by rational design and site-saturation mutagenesis. The ~67 kDa chimeric enzyme behaved as a monomer in solution, with a Km of 61 μM and a kcat of 15 s?1 for isocitrate in the presence of NADP+ and Mn2+. Our result demonstrated that dimeric IDHs have a potential to evolve monomeric ones. The evolution of the IDH family was also discussed.
- Tian, Changqing,Wen, Bin,Bian, Mingjie,Jin, Mingming,Wang, Peng,Xu, Lei,Zhu, Guoping
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p. 2396 - 2407
(2021/10/29)
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- Enhanced nonradical catalytic oxidation by encapsulating cobalt into nitrogen doped graphene: highlight on interfacial interactions
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Supported metal catalysts are widely used for heterogeneous catalytic processes (e.g., Fenton-like reaction), but the mechanisms of interfacial processes are still ambiguous. Herein, unique nanocarbon based catalysts with Co nanoparticles encapsulated in
- Yu, Xiaoyong,Wang, Lijing,Wang, Xin,Liu, Hongzhi,Wang, Ziyuan,Huang, Yixuan,Shan, Guoqiang,Wang, Weichao,Zhu, Lingyan
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supporting information
p. 7198 - 7207
(2021/03/29)
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- METHODS FOR IMPROVING YIELDS OF L-GLUFOSINATE
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Compositions and methods for the production of L-glufosinate are provided. The method involves converting racemic glufosinate to the L-glufosinate enantiomer or converting PRO to L-glufosinate in an efficient manner. In particular, the method involves the specific amination of PRO to L-glufosinate, using L-glutamate, racemic glutamate, or another amine source as an amine donor. PRO can be obtained by the oxidative deamination of D-glufosinate to PRO (2- oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid) or generated via chemical synthesis. PRO is then converted to L-glufosinate using a transaminase in the presence of an amine donor. When the amine donor donates an amine to PRO, L-glufosinate and a reaction by product are formed. Because the PRO remaining represents a yield loss of L-glufosinate, it is desirable to minimize the amount of PRO remaining in the reaction mixture. Degradation, other chemical modification, extraction, sequestration, binding, or other methods to reduce the effective concentration of the by-product, i.e., the corresponding alpha ketoacid or ketone to the chosen amine donor will shift the reaction equilibrium toward L-glufosinate, thereby reducing the amount of PRO and increasing the yield of L-glufosinate. Therefore, the methods described herein involve the conversion or elimination of the alpha ketoacid or ketone by-product to another product to shift the equilibrium towards L-glufosinate.
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Page/Page column 32
(2020/03/29)
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- The pseudoalteromonas luteoviolacea L-amino acid oxidase with antimicrobial activity is a flavoenzyme
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The marine environment is a rich source of antimicrobial compounds with promising pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of the highest proportions of bacterial species producing antimicrobial molecules. For decades, the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification, cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and shows an effective approach for the detection of novel LAAOs, whose study may be useful for biotechnological applications.
- Andreo-Vidal, Andrés,Sanchez-Amat, Antonio,Campillo-Brocal, Jonatan C.
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- Determinants of dual substrate specificity revealed by the crystal structure of homoisocitrate dehydrogenase from Thermus thermophilus in complex with homoisocitrate·Mg2+·NADH
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HICDH (Homoisocitrate dehydrogenase) is a member of the β-decarboxylating dehydrogenase family that catalyzes the conversion of homoisocitrate to α-ketoadipate using NAD+ as a coenzyme, which is the fourth reaction involved in lysine biosynthesis through the α-aminoadipate pathway. Although typical HICDHs from fungi and yeast exhibit strict substrate specificities toward homoisocitrate (HIC), HICDH from a thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both HIC and isocitrate (IC) as substrates at similar efficiencies. We herein determined the crystal structure of the quaternary complex of TtHICDH with HIC, NADH, and Mg2+ ion at a resolution of 2.5??. The structure revealed that the distal carboxyl group of HIC was recognized by the side chains of Ser72 and Arg85 from one subunit, and Asn173 from another subunit of a dimer unit. Model structures were constructed for TtHICDH in complex with IC and also for HICDH from Saccharomyces cerevisiae (ScHICDH) in complex with HIC. TtHICDH recognized the distal carboxyl group of IC by Arg85 in the model. In ScHICDH, the distal carboxyl group of HIC was recognized by the side chains of Ser98 and Ser108 from one subunit and Asn208 from another subunit of a dimer unit. By contrast, in ScHICDH, which lacks an Arg residue at the position corresponding to Arg85 in TtHICDH, these residues may not interact with the distal carboxyl group of shorter IC. These results provide a molecular basis for the differences in substrate specificities between TtHICDH and ScHICDH.
- Takahashi, Kento,Tomita, Takeo,Kuzuyama, Tomohisa,Nishiyama, Makoto
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p. 1688 - 1693
(2016/10/26)
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- Characterization of aromatic aminotransferases from Ephedra sinica Stapf
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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.
- Kilpatrick, Korey,Pajak, Agnieszka,Hagel, Jillian M.,Sumarah, Mark W.,Lewinsohn, Efraim,Facchini, Peter J.,Marsolais, Frédéric
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p. 1209 - 1220
(2016/04/26)
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- Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases by 2-Thiohydantoin Compounds
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Somatic mutations of isocitrate dehydrogenase 1 (IDH1) at R132 are frequently found in certain cancers such as glioma. With losing the activity of wild-type IDH1, the R132H and R132C mutant proteins can reduce α-ketoglutaric acid (α-KG) to d-2-hydroxyglutaric acid (D2HG). The resulting high concentration of D2HG inhibits many α-KG-dependent dioxygenases, including histone demethylases, to cause broad histone hypermethylation. These aberrant epigenetic changes are responsible for the initiation of these cancers. We report the synthesis, structure-activity relationships, enzyme kinetics, and binding thermodynamics of a novel series of 2-thiohydantoin and related compounds, among which several compounds are potent inhibitors of mutant IDH1 with Ki as low as 420 nM. X-ray crystal structures of IDH1(R132H) in complex with two inhibitors are reported, showing their inhibitor-protein interactions. These compounds can decrease the cellular concentration of D2HG, reduce the levels of histone methylation, and suppress the proliferation of stem-like cancer cells in BT142 glioma with IDH1 R132H mutation.
- Wu, Fangrui,Jiang, Hong,Zheng, Baisong,Kogiso, Mari,Yao, Yuan,Zhou, Chao,Li, Xiao-Nan,Song, Yongcheng
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p. 6899 - 6908
(2015/09/22)
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- Kinetic and mechanism of reactions of L-α-glutamic acid and L-Glutamine with pyridoxal
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The kinetics and mechanisms of condensation of pyridoxal with L-α-glutamic acid and L-glutamine were studied by UV spectroscopy and polarimetry. L-α-Glutamic acid reacts with pyridoxal to form a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and α-ketoglutaric acid. The reaction of Lglutamine with pyridoxal involves the Γ-NH 2 group and affords a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and L-α-glutamic acid.
- Pishchugin,Tuleberdiev
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p. 1362 - 1366
(2014/10/15)
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- Synthesis of L-2,3-diaminopropionic acid, a siderophore and antibiotic precursor
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L-2,3-diaminopropionic acid (L-Dap) is an amino acid that is a precursor of antibiotics and staphyloferrin B a siderophore produced by Staphylococcus aureus. SbnA and SbnB are encoded by the staphyloferrin B biosynthetic gene cluster and are implicated in L-Dap biosynthesis. We demonstrate here that SbnA uses PLP and substrates O-phospho-L-serine and L-glutamate to produce a metabolite N-(1-amino-1-carboxyl-2-ethyl)-glutamic acid (ACEGA). SbnB is shown to use NAD+ to oxidatively hydrolyze ACEGA to yield α-ketoglutarate and L-Dap. Also, we describe crystal structures of SbnB in complex with NADH and ACEGA as well as with NAD+ and α-ketoglutarate to reveal the residues required for substrate binding, oxidation, and hydrolysis. SbnA and SbnB contribute to the iron sparing response of S. aureus that enables staphyloferrin B biosynthesis in the absence of an active tricarboxylic acid cycle.
- Kobylarz, Marek J.,Grigg, Jason C.,Takayama, Shin-Ichi J.,Rai, Dushyant K.,Heinrichs, David E.,Murphy, Michael E.P.
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p. 379 - 388
(2014/04/03)
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- Meteorites as catalysts for prebiotic chemistry
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From outer space: Twelve meteorite specimens, representative of their major classes, catalyse the synthesis of nucleobases, carboxylic acids, aminoacids and low-molecular-weight compounds from formamide (see figure). Different chemical pathways are identified, the yields are high for a prebiotic process and the products come in rich and composite panels.
- Saladino, Raffaele,Botta, Giorgia,Delfino, Michela,Di Mauro, Ernesto
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p. 16916 - 16922
(2014/01/06)
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- Characterization of d-amino acid aminotransferase from Lactobacillus salivarius
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We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The Km values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.
- Kobayashi, Jyumpei,Shimizu, Yasuhiro,Mutaguchi, Yuta,Doi, Katsumi,Ohshima, Toshihisa
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- Metal-free oxidative synthesis of succinic acid from biomass-derived furan compounds using a solid acid catalyst with hydrogen peroxide
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Green oxidation of 2-furaldehyde (furfural) and its homologues were investigated using solid acid catalyst and hydrogen peroxide (H 2O2) in aqueous media to form succinic acid (SA), a key chemical that affords wide applications in macromolecular, pharmaceutical and agrochemical industries. Amberlyst-15, a strongly acidic ion-exchange resin, functioned as an efficient solid acid catalyst for a highly selective SA production in the furfural oxidation reaction. The synthesized SA was simply isolated in high purity (supported by NMR spectral data). Furthermore, the utilization of Amberlyst-15 with H2O2 was also useful in oxidizing carbonyl derivatives of furan (furfural, 5-hydroxymethyl-2- furaldehyde, furoic acid) to a one carbon less linear di-carboxylic acid such as SA and 2-oxoglutaric acid (OGA) under moderate conditions. This catalytic oxidation process with an acid catalyst and H2O2 promises to be a viable route for SA syntheses since they have the advantages of easy handling and simple purification decreasing the cost for the production, as compared to the current state of art.
- Choudhary, Hemant,Nishimura, Shun,Ebitani, Kohki
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- Biocatalytic asymmetric synthesis of unnatural amino acids through the cascade transfer of amino groups from primary amines onto keto acids
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Flee to the hills: An unfavorable equilibrium in the amino group transfer between amino acids and keto acids catalyzed by α-transaminases was successfully overcome by coupling with a ω-transaminase reaction as an equilibrium shifter, leading to efficient asymmetric synthesis of diverse unnatural amino acids, including L-tert-leucine and D-phenylglycine. Copyright
- Park, Eul-Soo,Dong, Joo-Young,Shin, Jong-Shik
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p. 3538 - 3542
(2014/01/06)
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- Arg305 of streptomyces l-glutamate oxidase plays a crucial role for substrate recognition
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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.
- Utsumi, Tomohiro,Arima, Jiro,Sakaguchi, Chika,Tamura, Takashi,Sasaki, Chiduko,Kusakabe, Hitoshi,Sugio, Shigetoshi,Inagaki, Kenji
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scheme or table
p. 951 - 955
(2012/06/29)
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- Photocatalytic reversible amination of α-keto acids on a ZnS surface: Implications for the prebiotic metabolism
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We report the enzyme-like reversible amination of four intermediates pertinent to the reductive tricarboxylic acid cycle on a photo-irradiated surface of mineral sphalerite (ZnS). Given its prevalence in the waters of early Earth, we suggest that the mineral-based photochemistry might have catalyzed the homeostasis of prebiotic metabolic systems.
- Wang, Wei,Li, Qiliang,Yang, Bin,Liu, Xiaoyang,Yang, Yanqiang,Su, Wenhui
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supporting information; experimental part
p. 2146 - 2148
(2012/03/26)
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- Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian D-aspartate oxidase
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d-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. Unlike DAO, which acts on several neutral and basic d-amino acids, DDO is highly specific for acidic d-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic d-amino acids, and showed a considerable decrease in activity against acidic d-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic d-amino acids and its activity against acidic d-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.
- Katane, Masumi,Saitoh, Yasuaki,Maeda, Kazuhiro,Hanai, Toshihiko,Sekine, Masae,Furuchi, Takemitsu,Homma, Hiroshi
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experimental part
p. 467 - 476
(2011/10/05)
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- PROCESS FOR STRAIGHTENING KERATIN FIBRES WITH A HEATING MEANS AND DENATURING AGENTS
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The invention relates to a process for straightening keratin fibres, comprising: (i) a step in which a straightening composition containing at least two denaturing agents is applied to the keratin fibres, (ii) a step in which the temperature of the keratin fibres is raised, using a heating means, to a temperature of between 110 and 250° C.
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- Branched-chain amino acid metabolon: Interaction of glutamate dehydrogenase with the mitochondrial branched-chain aminotransferase (BCATm)
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The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain α-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain α-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5′-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5′-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5′-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.
- Islam, Mohammad Mainul,Nautiyal, Manisha,Wynn, R. Max,Mobley, James A.,Chuang, David T.,Hutson, Susan M.
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experimental part
p. 265 - 276
(2010/12/24)
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- Stereospecific enzymatic transformation of α-ketoglutarate to (2S,3R)-3-methyl glutamate during acidic lipopeptide biosynthesis
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The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues Dptl from the daptomycin producer Streptomyces roseosporus and Lptl from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, Dptl, and Lptl are S-adenosyl methionine (SAM)-dependent α-ketoglutarate methyltransferases that catalyze the stereospecific methylation of α-ketoglutarate (αKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase IIvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of αKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of β-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes.
- Mahlert, Christoph,Kopp, Florian,Thirlway, Jenny,Micklefield, Jason,Marahiel, Mohamed A.
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p. 12011 - 12018
(2008/04/11)
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- ALPHA-KETOGLUTARATES AND THEIR USE AS THERAPEUTIC AGENTS
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The present invention relates generally to the field of pharmaceuticals and medicine. More particularly, the present invention relates to certain compounds (e.g., α-ketoglutarate compounds; compounds that activate HIFα hydroxylase; compounds that increases the level of α ketoglutarate, etc.) and their use in medicine, for example, in the treatment of cancer (e.g., cancer in which the activity of one of the enzymes in the tricarboxylic acid (TCA) cycle is down regulated), in the treatment of angiogenesis (e.g., hypoxia-induced angiogenesis). One preferred class of compounds are α-ketoglutarate compounds having a hydrophobic moiety that is, or is part of, an ester group formed from one of the acid groups of α ketogluartic acid; and pharmaceutically acceptable salts, solvates, amides, esters, ethers, N oxides, chemically protected forms, and prodrugs thereof.
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Page/Page column 60
(2008/06/13)
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- Hydrolysis of cyclopropane derivatives of aspartic and adipic acids
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Hydrolysis of substituted cyclopropane amino acids was performed. Free and partially substituted Z and E isomers of cyclopropane analogs of aspartic and adipic acids were obtained.
- Anisimova,Berkova,Deiko
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- Electrochemical incineration of 4-chlorophenol and the identification of products and intermediates by mass spectrometry
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This report summarizes results obtained as part of a larger effort to demonstrate the applicability of electrolytic procedures for the direct anodic (oxidative) degradation of toxic organic wastes. We refer to this process as "electrochemical incineration" (ECI) because the ultimate degradation products, e.g., carbon dioxide, are equivalent to those achieved by thermal incineration processes. In this work, the ECI of 4-chlorophenol is achieved in an aqueous medium using a platinum anode coated with a quaternary metal oxide film containing Ti, Ru, Sn, and Sb oxides. The electrode is stable and active when used with a solid Nafion membrane without the addition of soluble supporting electrolyte. Liquid chromatography (LC), including reverse phase and ion exchange chromatography, is coupled with electrospray mass spectrometry (ES-MS) and used, along with gas chromatography-mass spectrometry (GC-MS) and measurements of pH, chemical oxygen demand (COD), and total organic carbon (TOC), to study the reaction and identify the intermediate products from the ECI of 4-chlorophenol. Twenty-six intermediate products are identified and reported. The most abundant of these products are benzoquinone, 4-chlorocatechol, maleic acid, succinic acid, malonic acid, and the inorganic anions chloride, chlorate, and perchlorate. After 24 h of ECI, a solution that initially contained 108 ppm 4-chlorophenol yields only 1 ppm TOC with 98% of the original chlorine remaining in the specified inorganic forms. LC-ES-MS and direct infusion ES-MS detection limits are between 80 ppb and 4 ppm for these intermediate products. Elemental analysis of the electrolyzed solutions by inductively coupled plasma mass spectrometry ICP-MS showed that only trace amounts (25 ppb) of the metallic elements comprising the metal oxide film were present in the solution.
- Johnson, Steve K.,Houk, Linda L.,Feng, Jianren,Houk,Johnson, Dennis C.
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p. 2638 - 2644
(2007/10/03)
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- Production of D-glutamate from L-glutamate with glutamate racemase and L-glutamate oxidase
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We studied production of D-glutamate from L-glutamate using a bioreactor consisting of two columns of sequentially connected immobilized glutamate racemase (EC 5.1.1.3, from Bacillus subtilis IFO 3336) and L-glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was racemized by the glutamate racemase column, and then L-glutamate was oxidized by the L-glutamate oxidase column. Consequently only D-glutamate remained, and was easily separated from the α-ketoglutarate formed by anion-exchange chromatography. Both enzymes were highly stabilized by immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40°C) were similar to those of immobilized L-glutamate oxidase (pH 7, 50°C). Accordingly, we connected the two columns tandemly to do both enzyme reactions under the same conditions. Actually 4.5 μmol of D-glutamate was produced and isolated from 10 μmol of L-glutamate, about 90% of the theoretical yield.
- Oikawa, Tadao,Watanabe, Mayumi,Makiura, Hidemi,Kusakabe, Hitoshi,Yamade, Kazuhiro,Soda, Kenji
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p. 2168 - 2173
(2007/10/03)
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- An equilibrium and calorimetric study of some transamination reactions
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Apparent equilibrium constants and calorimetric enthalpies of reaction have been measured for the following enzyme-catalysed biochemical reactions at the temperature 298.15 K: L-alanine(aq) + 2-oxoglutarate(aq) = pyruvate(aq) + L-glutamate(aq); L-tyrosine(aq) + 2-oxoglutarate(aq) = 4-hydroxyphenylpyruvate(aq) + L-glutamate(aq); and L-phenylalanine(aq) + 2-oxoglutarate(aq) = phenylpyruvate(aq) + L-glutamate(aq). The results are used to calculate equilibrium constants and standard molar enthalpy, entropy, and Gibbs energy changes for reference reactions involving specific species. Apparent equilibrium constants and standard transformed Gibbs energy changes for these reactions under physiological conditions have also been calculated. The results are discussed in terms of the changes in chemical bonding characteristic of transamination reactions.
- Tewari, Yadu B.,Kishore, Nand,Goldberg, Robert N.,Luong, Tinh N.
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p. 777 - 793
(2007/10/03)
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- EFFECT OF HALOGEN SUBSTITUTION OF INDOLE-3-ACETIC ACID ON BIOLOGICAL ACTIVITY IN PEA FRUIT
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Auxins (a class of plant growth hormones naturally present in all plants) have been implicated in fruit growth of pea.Pea (Pisum sativum L.) fruit contain the auxins indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA).Fruits grow poorly and subsequently abscise when seeds are removed two days after anthesis, but 4-Cl-IAA can substitute for the seeds in maintaining growth of deseeded fruit (perciarp) in planta.Applications of 4-Cl-IAA promoted pericarp growth, the effect increasing with concentration from 1 to 100 μM, but IAA was ineffective in stimulating growth when tested from 0.1 to 100 μM.The effect of the position of the halogen on pericarp growth was examined by assaying the activities of 4-, 5-, 6- and 7-chloro- and fluoro-substituted IAA.The position and type of halogen dramatically affected auxin activity, with the natural product 4-Cl-IAA being most effective.Of the other compounds tested, only 5-Cl-IAA stimulated pea pericarp elongation, and then only moderately.Fluoro-substituted IAAs did not stimulate pericarp growth, and 4-F-IAA was inhibitory.This study is unique in that it reports the biological activity of 4-Cl-IAA and halogen-IAA analogues in tissues of intact plants known to contain 4-Cl-IAA.The relative activity of the compounds is discussed in reference to previous reports of auxin activity in other systems, and 4-Cl-IAA's possible importance in pea fruit growth. - Key words: Pisum sativum; Leguminosae; pea pericarp; auxin; IAA; 4-chloroindole-3-acetic acid; helogenated-IAA analogues.
- Reinecke, Dennis M.,Ozga, Jocelyn A.,Magnus, Volker
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p. 1361 - 1366
(2007/10/02)
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- POLAROGRAPHIC AND POTENTIOMETRIC INVESTIGATION OF 2-OXOGLUTARIC ACID
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The first-stage and second-stage concentrational and thermodynamic acid dissociation constants of 2-oxoglutaric acid, and also the equilibrium constants for the hydration of its molecules and doubly charged anions, were determined.The kinetic polarographic current of 2-oxoglutaric acid arises from the slow recombination of the dianion, whereas the stage of the dehydration of the dianion is fairly fast.The rate constant for the recombination of the dianion of 2-oxoglutaric acid with the hydroxonium ion was determined.
- Kunchev, K. V.,Tur'yan, Ya. I.,Dinkov, Kh. A.
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p. 311 - 315
(2007/10/02)
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- Chemo-enzymatic synthesis of specifically stable-isotope labelled L-glutamic acid and 2-oxoglutaric acid
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(3-13C)-(4-13C)-,(5-13C)- and (3,4-13C2)-2-oxoglutaric acid were prepared starting from the simple 13C-enriched compounds: ethyl bromoacetate and paraformaldehyde, via a single reaction scheme on the gram scale in high yield.This reaction scheme allows specific 13C enrichment of every carbon position and any combination of positions. (3-13C)-,(4-13C)-, (5-13C), (3,4-13C2) and (15N)-L-glutamic acid were prepared by converting the corresponding 2-oxoglutaric acids via an enantioselective enzymatic conversion.The labelled L-glutamic acids and 2-oxoglutaric acids were characterized by 1H NMR, 13C NMR and mass spectrometry.
- Cappon, J. J.,Baart, J.,Walle, G. A. M. van der,Raap, J.,Lugtenburg, J.
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p. 158 - 166
(2007/10/02)
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- Tryptophan Production by a Lipoic Acid Auxotroph of Enterobacter aerogenes Having Both Pyruvic Acid Productivity and High Tryptophanase Activity
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A new process for tryptophan production was established using a lipoic acid auxotrophic mutant, Enterobacter aerogenes L-12, which has both pyruvic acid productivity and tryptophanase activity.The process consists of the production of pyruvic acid from glucose by the washed cells and the subsequent conversion of the acid to tryptophan by the tryptophanase itself in the presence of indole and NH4Cl.To prepare washed cells of which the tryptophanase activity and the pyruvic acid productivity were both high, it was best to culture the strain in a medium containing 1percent Polypepton, 0.2percent glucose, 3μg/l DL-lipoic acid, 0.05percent L-tryptophan, and mineral salts.The optimum composition of the reaction mixture for the pyruvic acid production by the washed cells was established.Under these conditions, 17 g/l of pyruvic acid was accumulated from 5percent glucose after 36 hr of incubation.Thus, the conversion of the pyruvic acid to tryptophan was done by adding indole, NH4Cl, pyridoxal-5'-phosphate, Triton X-100, and KOH to adjust the pH to 9.0 to the above reaction mixture.As a result, the pyruvic acid was rapidly converted to tryptophan, and the concentration of 14 g/l was obtained after 36 hr (total 72 hr).
- Yokota, Atsushi,Oita, Shigeru,Takao, Shoichi
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p. 2037 - 2044
(2007/10/02)
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- CHEMISTRY OF A NEW ANTIBIOTIC: LACTIVICIN
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A novel antibiotic, lactivicin (LTV), was isolated from the culture filtrates of two bacterial strains by various types of chromatography.LTV exists in aqueous solution as an equilibrium mixture of two epimers in a ratio of about 1:1.The chemical structure of LTV (C10H12N2O7) was determined to be 2-(4S-acetylamino-3-oxo-2-isoxazolidinyl)-5-oxo-tetrahydrofuran-2-carboxylic acid.The absolute configuration at the C-6 position was elucidated from the CD spectral data and X-ray crystallographic analysis of 4-amino-lactivinic acid obtained by the iminoether method.This compound is useful as a starting material for chemical modification.LTV showed antibacterial activity against Gram-positive and negative bacteria, susceptibility to β-lactamases, and affinity for penicillin-binding proteins.We therefore concluded that LTV is a novel skeleton antibiotic having β-lactam-like activities.
- Harada, Setsuo,Tsubotani, Shigetoshi,Hida, Tsuneaki,Koyama, Katsuo,Kondo, Masahiro,Ono, Hideo
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p. 6589 - 6606
(2007/10/02)
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- Preparation having excellent absorption property
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A preparation containing an absorption promoter selected from specific classes of water-soluble macromolecular compounds having chelating activity, preferably in the presence of a salt at a concentration such that the composition exhibits higher osmotic pressure than isotonic sodium chloride solution, and a medicine is found to promote absorption of the medicine through a gastrointestinal organ such as the colon, rectum, or vagina.
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- STRUCTURE OF LACTIVICIN, AN ANTIBIOTIC HAVING A NEW NUCLEUS AND SIMILAR BIOLOGICAL ACTIVITIES TO β-LACTAM ANTIBIOTICS
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The structure of a new antibiotic, lactivicin, was determined to be -2-(4-acetylamino-3-oxo-2-isoxazolidinyl)-5-oxo-tetrahydrofuran-2-carboxylic acid.
- Harada, Setsuo,Tsubotani, Shigetoshi,Hida, Tsuneaki,Ono, Hideo,Okazaki, Hisayoshi
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p. 6229 - 6232
(2007/10/02)
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- Studies on Fermantation Products and Metabolism of Long Chain Alkyl Diamine in Microorganisms
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Ten bacteria capable of using 1,12-diaminododecane (DAD) as a sole carbon and energy source were isolated from soil samples.Four strains (K95, K55, K24, 110-2)were identified as Pseudomonas, four (DAD2-3, 10-23-A, 10-23-B, K61) as Nocardia and two (DAD2-1, 1994) as Corynebacterium on the basis of their taxonomic characteristics.Metabolic products such as 12-aminododecanoic acid, 12-hydroxydodecanoic acid, 1,10-decanedicarboxylic acid, sebacic acid, suberic acid, adipic acid and α-ketoglutaric acid were found in the culture broths of various bacteria.The yields of 12-aminododecanoic acid and 12-hydroxydodecanoic acid from DAD were as high as 55 percent and 20 percent, respectively.An amine dehydrogenase was found in cell-free extracts of Pseudomonas K95 and amine oxydases were detected in cell-free extracts of Nocardia 10-23-A.A biodegradation pathway for DAD is proposed.
- Niimura, Youichi,Yinbo, Qu,Lee, Sang-Joon,Omori, Toshio,Minoda, Yasuji
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p. 1437 - 1444
(2007/10/02)
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- Meerwein Reduction of Levulinic Acid Derivatives: A New Route for the Synthesis of Glutamic Acid
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Glutamic acid has been prepared from levulinic acid by a new route.In this method α-ketoglutaric (III) acid is obtained by oxidation of δ-benzallevulinic acid (II) and converted into diethyl ester (IV) which on Meerwein-Pondorff-Verley reduction furnishes the hydroxy derivative (V).The latter (V) is converted into chloro derivative (VI) which on amination gives glutamic acid (VII) in a good yield.Meerwein-Pondorff-Verley reduction of ethyl levulinate, ethyl β-bromolevulinate and 2,7-octanedione has also been studied.However, such a reduction of ethyl β-aminolevulinate remains unsuccessfull.
- Joshi, Uday R.,Limaye, P. A.
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p. 1176 - 1178
(2007/10/02)
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- Synthesis and Properties of Some 7-Dimethylamino-1,4-benzoxazin-2-ones
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The synthesis of the 7-dimethylamino-1,4-benzoxazin-2-ones (5), fluorescent dyes, by condensing α-ketoacids with 2-amino-5-dimethylaminophenol is described.When the 3-substituent is a methyl group, these compounds can be further condensed with aromatic aldehydes to provide the styryl dyes (6).These products are easily opened by hydrochloric acid in ethanolic solution to afford the corresponding benzalketoacid ethyl esters (7).
- Bris, Marie-Therese
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p. 1275 - 1280
(2007/10/02)
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- Extracellular Production of D-(+)-2-Hydroxyglutaric Acid by Yarrowia lipilytica from Glucose under Aerobic, Thiamine-deficient Conditions
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Under thiamine-deficient, aerobic culture conditions, Yarrowia lipolytica was found to produce D-(+)-2-hydroxyglutaric acid extracellularly in amounts of about 5 mg per ml of the culture filtrate, together with pyruvic and 2-ketoglutaric acids, from glucose or glycerol in a chemically defined medium.Under similar conditions, however, only a small amount of the hydroxy acid was produced from odd- or even-numbered n-alkanes.
- Oogaki, Masako,Nakahara, Tadaatsu,Uchiyama, Hiroo,Tabuchi, Takeshi
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p. 2619 - 2624
(2007/10/02)
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- L-Glutamate Oxidase from Streptomyces violascens. II. Properties
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The properties of L-glutamate oxidase, a novel L-glutamic acid oxidizing enzyme from Streptomyces violascens H82-N-SY7 were examined.The enzyme showed absorption maxima at 280, 390 and 470 nm and a marked shoulder at 490 nm, and contained 1 mol of flavin-adenine dinucleotide (FAD) per mol of enzyme.Enzyme activity was inhibited by Ag+, Hg2+, p-chloromercuribenzoate (PCMB) and N-bromosuccinimide.The enzyme catalyzed the oxidation of L-glutamic acid using molecular oxygen as a primary electron acceptor and produced α-ketoglutaric acid, NH3 and hydrogen peroxide according to the following schema: L-glutamic acid+H2O+O2->α-ketoglutaric acid+NH3+H2O2.L-Glutamate oxidase was used in a specific and sensitive determination procedure for L-glutamic acid.Keywords - L-glutamate oxidase; Streptomyces violascens; flavoprotein; enzymatic determination of L-glutamic acid
- Kamei, Toshio,Asano, Kazuko,Kondo, Hisao,Matsuzaki, Meiki,Nakamura, Shoshiro
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p. 3609 - 3616
(2007/10/02)
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- Purification and Properties of a New Enzyme, L-Glutamate Oxidase, from Streptomyces sp. X-119-6 Grown on Wheat Bran
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A new flavoprotein enzyme, L-glutamate oxidase, was purified to homogeneity from an aqueous extract of a wheat bran culture of Streptomyces sp.X-119-6.It showed absorption maxima at 273, 385 and 465 nm and a shoulder around 490 nm, and contained 2 mol of FAD per mol of enzyme.The enzyme had a molecular weight of approximately 140,000 and consisted of three sizes of subunits with molecular weights of 44,000, 16,000 and 9,000.Balance studies showed that 1 mol of L-glutamate was converted to 1 mol of α-ketoglutarate, ammonia and hydrogen peroxide with the consumption of 1 mol of oxygen.In addition to L-glutamate, L-aspartate was oxidized by the enzyme but only to an extent of 0.6percent at pH 7.4; the Michaelis constants were as follows: 0.21 mM for L-glutamate and 29 mM for L-aspartate.The isoelectric point was pH 6.2, and the enzyme activity was optimal between pH 7.0 and 8.0.When the enzyme was heated at pH 5.5 for 15 min, the remaining activity was 100percent of the original activity level at 65 deg C, 87percent at 75 deg C and 47percent at 85 deg C.
- Kusakabe, Hitoshi,Midorikawa, Yuichiro,Fujishima, Tetsuro,Kuninaka, Akira,Yoshino, Hiroshi
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p. 1323 - 1328
(2007/10/02)
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