- 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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- Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+-Binding Photoprotein Aequorin
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Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization–time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca2+, similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.
- Hosoya, Takamitsu,Inouye, Satoshi,Nakamura, Mitsuhiro
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- Butenolide derivative as well as preparation method and application thereof
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The invention discloses a butenolide compound as well as a preparation method and an application thereof. The butenolide derivative has the inhibitory activity of protein tyrosine phosphatase 1B (PTP1B), improves insulin resistance of HepG2 cells, generates a remarkable hypoglycemic effect and can be used for preparing a medicine for treating diabetes mellitus.
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- Biocascade Synthesis of L-Tyrosine Derivatives by Coupling a Thermophilic Tyrosine Phenol-Lyase and L-Lactate Oxidase
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A one-pot biocascade of two enzymatic steps catalyzed by an l-lactate oxidase and a tyrosine phenol-lyase has been successfully developed in the present study. The reaction provides an efficient method for the synthesis of l-tyrosine derivatives, which exhibits readily available starting materials and excellent yields. In the first step, an in situ generation of pyruvate from readily available bio-based l-lactate catalyzed by a highly active l-lactate oxidase from Aerococcus viridans (AvLOX) was developed (using oxygen as oxidant and catalase as hydrogen peroxide removing reagent). Pyruvate thus produced underwent C–C coupling with phenol derivatives as acceptor substrate using specially designed thermophilic tyrosine phenol-lyase mutants from Symbiobacterium toebii (TTPL). Overall, this cascade avoids the high cost and easy decomposition of pyruvate and offered an efficient and environmentally friendly procedure for l-tyrosine derivatives synthesis.
- Jiang, Yiqi,Ju, Shuyun,Li, Guosi,Lian, Jiazhang,Lin, Jianping,Wu, Mianbin,Xue, Hailong,Yang, Lirong
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supporting information
(2020/02/25)
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- Efficient Synthesis of Phenylacetate and 2-Phenylethanol by Modular Cascade Biocatalysis
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The green and sustainable synthesis of chemicals from renewable feedstocks by a biotransformation approach has gained increasing attention in recent years. In this work, we developed enzymatic cascades to efficiently convert l-phenylalanine into 2-phenylethanol (2-PE) and phenylacetic acid (PAA), l-tyrosine into tyrosol (p-hydroxyphenylethanol, p-HPE) and p-hydroxyphenylacetic acid (p-HPAA). The enzymatic cascade was cast into an aromatic aldehyde formation module, followed by an aldehyde reduction module, or aldehyde oxidation module, to achieve one-pot biotransformation by using recombinant Escherichia coli. Biotransformation of 50 mM l-Phe produced 6.76 g/L PAA with more than 99 % conversion and 5.95 g/L of 2-PE with 97 % conversion. The bioconversion efficiencies of p-HPAA and p-HPE from l-Tyr reached to 88 and 94 %, respectively. In addition, m-fluoro-phenylalanine was further employed as an unnatural aromatic amino acid substrate to obtain m-fluoro-phenylacetic acid; '96 % conversion was achieved. Our results thus demonstrated high-yielding and potential industrial synthesis of above aromatic compounds by one-pot cascade biocatalysis.
- Mao, Zuoxi,Liu, Lijun,Zhang, Yang,Yuan, Jifeng
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p. 2676 - 2679
(2020/06/03)
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- Scope and limitations of reductive amination catalyzed by half-sandwich iridium complexes under mild reaction conditions
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The conversion of aldehydes and ketones to 1° amines could be promoted by half-sandwich iridium complexes using ammonium formate as both the nitrogen and hydride source. To optimize this method for green chemical synthesis, we tested various carbonyl substrates in common polar solvents at physiological temperature (37 °C) and ambient pressure. We found that in methanol, excellent selectivity for the amine over alcohol/amide products could be achieved for a broad assortment of carbonyl-containing compounds. In aqueous media, selective reduction of carbonyls to 1° amines was achieved in the absence of acids. Unfortunately, at Ir catalyst concentrations of 1 mM in water, reductive amination efficiency dropped significantly, which suggest that this catalytic methodology might be not suitable for aqueous applications where very low catalyst concentration is required (e.g., inside living cells).
- Nguyen, Dat P.,Sladek, Rudolph N.,Do, Loi H.
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supporting information
(2020/07/15)
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- Carbonic Anhydrase XII Inhibitors Overcome Temozolomide Resistance in Glioblastoma
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The natural product primary sulfonamide, psammaplin C (1), when used in combination with clinically used chemotherapeutic drugs, including temozolomide, reverses multidrug resistance and increases survival in glioblastoma, a highly aggressive primary brain tumor. We showed previously that the mechanism of action of 1 is novel, acting to indirectly interfere with P-glycoprotein drug efflux activity as a consequence of carbonic anhydrase XII (CA XII) inhibition. To build structure-activity relationships, 45 derivatives of 1 were designed, synthesized, and evaluated against a panel of CA isoforms. Compound 55 was identified as a potent inhibitor of CA XII (Ki = 0.56 nM) and was investigated in vitro and in vivo using samples from glioblastoma patients. The results strengthen the possibility that co-therapy of temozolomide with a CA XII inhibitor may more effectively treat glioblastoma by suppressing an important temozolomide resistance mechanism.
- Mujumdar, Prashant,Kopecka, Joanna,Bua, Silvia,Supuran, Claudiu T.,Riganti, Chiara,Poulsen, Sally-Ann
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p. 4174 - 4192
(2019/05/01)
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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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- Bio-inspired enantioselective full transamination using readily available cyclodextrin
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The mimics of vitamin B6-dependent enzymes that catalyzed an enantioselective full transamination in the pure aqueous phase have been realized for the first time through the establishment of a new “pyridoxal 5′-phosphate (PLP) catalyzed non-covalent cyclodextrin (CD)-keto acid inclusion complexes” system, and various optically active amino acids have been obtained.
- Zhang, Shiqi,Li, Guangxun,Liu, Hongxin,Wang, Yingwei,Cao, Yuan,Zhao, Gang,Tang, Zhuo
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p. 4203 - 4208
(2017/02/05)
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- Synthesis, biological evaluation and molecular modeling studies of psammaplin A and its analogs as potent histone deacetylases inhibitors and cytotoxic agents
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In this study, a concise synthetic method of psammaplin A was achieved from 3-bromo-4-hydroxybenzaldahyde and hydantoin through a four-step synthesis via Knoevenagel condensation, hydrolysis, oximation and amidation in 37% overall yield. A collection of novel psammaplin A analogs focused on the variations of substituents at the benzene ring and modifications at the oxime moiety were synthesized. Among all the synthesized compounds, 5d and 5e showed better HDAC inhibition than psammaplin A and comparable cytotoxicity against four cancer cell lines (PC-3, MCF-7, A549 and HL-60). Molecular docking and dynamics simulation revealed that (i) hydrogen atom of the oxime group interacts with Asp99 of HDAC1 through a water bridged hydrogen bond and (ii) a hydroxyl group is optimal attached on the para-position of benzene, interacting with Glu203 at the entrance to the active site tunnel.
- Wen, Jiachen,Bao, Yu,Niu, Qun,Liu, Jiang,Yang, Jinyu,Wang, Wanqiao,Jiang, Tao,Fan, Yinbo,Li, Kun,Wang, Jian,Zhao, Linxiang,Liu, Dan
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supporting information
p. 4372 - 4376
(2016/08/18)
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- Compositions and methods for producing benzylisoquinoline alkaloids
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The present invention relates to host cells that produce compounds that are characterized as benzylisoquinolines, as well as select precursors and intermediates thereof. The host cells comprise one, two or more heterologous coding sequences wherein each of the heterologous coding sequences encodes an enzyme involved in the metabolic pathway of a benzylisoquinoline, or its precursors or intermediates from a starting compound. The invention also relates to methods of producing the benzylisoquinoline, as well as select precursors and intermediates thereof by culturing the host cells under culture conditions that promote expression of the enzymes that produce the benzylisoquinoline or precursors or intermediates thereof.
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Page/Page column 4
(2016/05/19)
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- Mechanistic study of the radical SAM-dependent amine dehydrogenation reactions
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The radical SAM enzyme NosL catalyzes the conversion of l-Trp to 3-methyl-2-indolic acid, and this reaction is initiated by the 5′-deoxyadenosyl (dAdo) radical-mediated hydrogen abstraction from the l-Trp amino group. We demonstrate here that when d-Trp was used in the NosL reaction, hydrogen abstraction occurs promiscuously at both the amino group and Cα of d-Trp. These results inspired us to establish the detailed mechanism of l-Trp amine dehydrogenation catalyzed by a NosL mutant, and to engineer a novel radical SAM-dependent l-Tyr amine dehydrogenase from the thiamine biosynthesis enzyme ThiH.
- Ji, Xinjian,Liu, Wan-Qiu,Yuan, Shuguang,Yin, Yue,Ding, Wei,Zhang, Qi
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p. 10555 - 10558
(2016/09/02)
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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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- Biocontrolled formal inversion or retention of L -α-amino acids to enantiopure (R)- or (S)-hydroxyacids
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Natural L-α-amino acids and L-norleucine were transformed to the corresponding α-hydroxy acids by formal biocatalytic inversion or retention of absolute configuration. The one-pot transformation was achieved by a concurrent oxidation reduction cascade in aqueous media. A representative panel of enantiopure (R)- and (S)-2-hydroxy acids possessing aliphatic, aromatic and heteroaromatic moieties were isolated in high yield (67-85 %) and enantiopure form (>99 % ee) without requiring chromatographic purification.
- Busto, Eduardo,Grischek, Barbara,Kroutil, Wolfgang,Richter, Nina
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supporting information
p. 11225 - 11228,4
(2015/01/07)
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- Isolation, purification, and characterization of phenylpyruvate transaminating enzymes of Erwinia carotovora
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Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively.
- Paloyan,Hambardzumyan,Halebyan
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scheme or table
p. 98 - 104
(2012/06/29)
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- Mutant d-amino acid oxidase with higher catalytic efficiency toward d-amino acids with bulky side chains
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d-Amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO) is widely used in fine organic synthesis, including the preparation of unnatural l-amino acids and α-keto acids. The analysis of the three-dimensional structure of TvDAAO was carried out with the aim of producing the enzyme specific to d-amino acids with bulky side chains. The analysis revealed the residue Phe54 at the entrance to the active site, which controls the substrate access to this site. The residue Phe54 was replaced by residues Ala, Ser, and Tyr. The cultivation of recombinant E. coli strains expressing TvDAAO mutants showed that the mutein with the Phe54Ala substitution had very low stability. Thus, the inactivation of the enzyme occured within 10 min after the cell disruption. The Phe54Ser TvDAAO and Phe54Tyr TvDAAO mutants were obtained as homogeneous preparations, and their thermal stability and catalytic properties were investigated. The introduction of Phe54Ser and Phe54Tyr substitutions resulted in additional stabilization of the protein macromolecule compared to the wild-type TvDAAO. Thus, the half-inactivation time for the mutant enzymes at 54 C increased by a factor of 1.5 and 2, respectively. As in the case of wild-type TvDAAO, the thermal inactivation of the muteins proceeds via a two-step dissociative mechanism. The introduction of mutations led to a strong change in the substrate specificity profile. The mutants have no activity toward a series of d-amino acids (Phe54Ser TvDAAO toward d-Ala, d-Ser, d-Val, and d-Thr; Phe54Tyr TvDAAO toward d-Ser, d-Tyr, d-Thr, and d-Lys). The catalytic efficiency (the k cat/K M ratio) of the Phe54Ser TvDAAO mutant toward d-amino acids with bulky side chains (d-Lys, d-Asn, d-Phe, d-Tyr, d-Trp, and d-Leu) increased from 2.4 to 7.3 times.
- Komarova,Golubev,Khoronenkova,Tishkov
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p. 1489 - 1496
(2013/11/19)
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- The role of residues Arg169 and Arg220 in intersubunit interactions of yeast D-Amino acid oxidase
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D-Amino acid oxidase from the yeast Trigonopsis variabilis (EC 1.4.3.3, TvDAAO) exists as a dimer consisting of two identical subunits. The dimeric structure of the enzyme is stabilized by 12 (six pairs) hydrogen bonds, the residues Arg169 and Arg220 of each subunit being involved in eight hydrogen bonds. The Arg169Glu and Arg(169,220)Ala mutants of TvDAAO were prepared. Both mutant enzymes were expressed in E. coli cells as insoluble but catalytically active inclusion bodies. The introduction of amino acid substitutions at the intersubunit interface resulted in a change in the substrate specificity profile and a strong decrease in thermal stability.
- Cherskova,Khoronenkova,Tishkov
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scheme or table
p. 269 - 275
(2011/01/06)
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- Inactivation of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor by 2-oxo-4-phenyl-3-butynoate
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Macrophage migration inhibitory factor (MIF) is an important immunoregulatory protein that has been implicated in several inflammatory diseases. MIF also has a phenylpyruvate tautomerase (PPT) activity, the role of which remains elusive in these biological activities. The acetylene compound, 2-oxo-4-phenyl-3-butynoate (2-OPB), has been synthesized and tested as a potential irreversible inhibitor of its enzymatic activity. Incubation of the compound with MIF results in the rapid and irreversible loss of the PPT activity. Mass spectral analysis established that the amino-terminal proline, previously implicated as a catalytic base in the PPT-catalyzed reaction, is the site of covalent modification. Inactivation of the PPT activity likely occurs by a Michael addition of Pro-1 to C-4 of the inhibitor. Attempts to crystallize the inactivated complex to confirm the structure of the adduct on the covalently modified Pro-1 by X-ray crystallography were not successful. Nor was it possible to unambiguously interpret electron density observed in the active sites of the native crystals soaked with the inhibitor. This may be due to crystal packing in that the side chain of Glu-16 from an adjacent trimer occupies one active site. However, this crystal contact may be partially responsible for the high-resolution quality of these MIF crystals. Nonetheless, because MIF is a member of the tautomerase superfamily, a group of structurally homologous proteins that share a β-α-β structural motif and a catalytic Pro-1, 2-OPB may find general use as a probe of tautomerase superfamily members that function as PPTs.
- Golubkov, Pavel A.,Johnson Jr., William H.,Czerwinski, Robert M.,Person, Maria D.,Wang, Susan C.,Whitman, Christian P.,Hackert, Marvin L.
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p. 183 - 199
(2008/02/09)
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- Process for the preparation of new antidiabetic agents
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The present invention relates to an improved process for the preparation of novel antidiabetic compounds having formula (1) where R1represents hydrogen or lower alkyl group and X represents hydrogen or halogen atom.
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- Combinatorial synthesis through disulfide exchange: Discovery of potent psammaplin A type antibacterial agents active against methicillin-resistant Staphylococcus aureus (MRSA)
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Psammaplin A is a symmetrical bromotyrosine-derived disulfide natural product isolated from the Psammaplysilla sponge, which exhibits in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Inspired by the structure of this marine natural product, a combinatorial scrambling strategy for the construction of heterodimeric disulfide analogues was developed and applied to the construction of a 3828-membered library starting from 88 homodimeric disulfides. These psammaplin A analogues were screened directly against various gram positive bacterial strains leading to the discovery of a series of potent antibacterial agents active against methicillin-resistant Staphylococcus aureus (MRSA). Among the most active leads derived from these studies are compounds 104, 105, 113, 115, 123, and 128. The present, catalytically-induced, disulfide exchange strategy may be extendable to other types of building blocks bearing thiol groups facilitating the construction of diverse discovery-oriented combinatorial libraries.
- Nicolaou,Hughes, Robert,Pfefferkorn, Jeffrey A.,Barluenga, Sofia,Roecker
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p. 4280 - 4295
(2007/10/03)
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- Chemical studies on the nyctinastic leaf-movement regulated by a biological clock
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Most leguminosae plants close their leaves in the evening as if to sleep, and open them early in the morning. This circadian rhythm is known to be controlled by the biological clock of such plants. Extensive studies on other nyctinastic plants led to the isolation of a variety of leaf-closing and leaf-opening substances. Based on our experiments on these plants is controlled by a biological clock that regulates the balance of concentration between leaf-opening and -closing substances.
- Ueda, Minoru,Yamamura, Shosuke
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p. 927 - 940
(2007/10/03)
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- Process for production of phenyllactic acid derivative
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A process for producing a phenyllactic acid derivative comprises hydrogenating a phenylpyruvic acid derivative in the presence of a catalyst containing at least one element selected from the Group VIII elements of the periodic table. The process provides a phenyllactic acid derivative useful as an intermediate in the production of pharmaceuticals and agricultural chemicals. The process uses as a starting material a phenylpyruvic acid derivative which is easily synthesized. The process is simpler in operation and provides a higher yield than conventional processes.
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