Enzyme self-inactivation is a main limitation of the preparation of eicosanoids

Synthesis of prostanoids is accompanied by various processes reducing the product yield. These processes are also known to affect syntheses of thromboxane (TX) and 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-HETE). Partially purified preparations of TX synthase and prostaglandin (PG) synthase were used to optimize TX synthesis with respect to concentrations of the enzymes and eicosapolyenoic acid (EPA). Conditions for the maximum product yield and the minimum consumption of enzymes were determined. Consumption of the TX synthase was large owing to its inactivation during the reaction and the nonenzymatic destruction of the intermediate product PG-endoperoxide. Separate addition of PG and TX synthases increased the product yield by preventing EPA sorption on ballast proteins. Microsomal 12-lipoxygenase (12-LO) was also shown to be inactivated during the reaction, and this process was the major limitation of 12-HETE synthesis. Lipoxygenase reaction in the presence of some reducing agents led to a considerable increase of the 12-HETE yield, supposedly by preventing further oxidation of the 12-LO reaction product 12-hydroperoxy derivative of eicosatetraenoic acid. The possibility of using human blood platelet microsomes for preparation of some derivatives of EPAs is discussed.     

The influence of additives on the X-ray induced aggregation of malate synthase monitoring of the aggregation processin situ by time-resolved small-angle X-ray scattering

The X-ray induced aggregation of the sulfhydryl enzyme malate synthase in aqueous solution was monitoredin situ by time-resolved small-angle X-ray scattering. Experiments were performed in the absence/presence of various additives: formate, superoxide dismutase, catalase, NaCl, acetyl-CoA, glyoxylate, malate, pyruvate, -ketobutyrate, oxaloacetate, glycollate, lactate. The scattering curves were measured as a function of the time of irradiation and were analysed in terms of radii of gyration, degrees of aggregation, distance distribution functions, and parameters derived therefrom. Irradiation in the absence of additives resulted in a strong aggregation of enzyme particles. Each of the additives impeded aggregation, however to a different extent. The OH scavenger formate reduced aggregation efficiently; less pronounced effects were registered for superoxide dismutase and/or catalase (the scavengers for and H₂O₂), and for NaCl. Very pronounced diminutions of the aggregation phenomena were provided by substrates or analogues; the efficiency of these substances as radioprotectors may be explained by their action as both scavengers and specific ligands. Based on these results some implications for the performance of conventional small-angle X-ray scattering experiments on biopolymers are derived.

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Der Einfluß von Zusätzen auf die röntgeninduzierte Aggregation der Malatsynthase. Registrierung des Aggregationsvorganges in situ mittels zeitaufgelöster Röntgenkleinwinkelstreuung

Zusammenfassung Die röntgeninduzierte Aggregation des Sulfhydrylenzyms Malatsynthase in wäßriger Lösung wurdein situ mittels zeitaufgelöster Röntgenkleinwinkelstreuung messend verfolgt. Streuexperimente wurden in An- bzw. Abwesenheit verschiedener Zusätze durchgeführt: Formiat, Superoxiddismutase, Catalase, NaCl, Acetyl-CoA, Glyoxylat, Malat, Pyruvat, -Ketobutyrat, Oxalacetat, Glycolat, Lactat. Die Streukurven wurden als Funktion der Bestrahlungsdauer registriert. Die Auswertung lieferte Streumassenradien, Aggregationsgrade, Abstandsverteilungsfunktionen, und daraus abgeleitete Parameter. Die Bestrahlung in Abwesenheit von Zusätzen verursachte eine starke Aggregation der Enzymteilchen. Jeder der Zusätze verminderte die Aggregation, wenn auch in unterschiedlichem Ausmaß. Der OH Fänger Formiat verringerte die Aggregation wirksam; weniger stark ausgeprägte Effekte ergaben sich für die bzw. H₂O₂ Fänger Superoxiddismutase bzw. Catalase und für NaCl. Substrate und Substratanaloge reduzierten das Ausmaß der Aggregation besonders wirkungsvoll; der Schutzeffekt dieser Substanzen kann durch ihre zweifache Wirkung als Fänger bzw. spezifische Liganden erklärt werden. Ausgehend von diesen Ergebnissen werden einige Schlußfolgerungen für die Durchführung üblicher Röntgenkleinwinkelexperimente an Biopolymeren abgeleitet.

     

Insights into the Dual Activity of a Bifunctional Dehydratase‐Cyclase Domain

Oxygen‐containing heterocycles are a common structural motif in polyketide natural products and contribute significantly to their biological activity. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratase (DH) and pyran‐forming cyclase in ambruticin biosynthesis. AmbDH3 is similar to monofunctional DH domains, using H51 and D215 for dehydration. V173 was confirmed as a diagnostic residue for cyclization activity by a mutational study and enzymatic in vitro experiments. Similar motifs were observed in the seemingly monofunctional AmbDH2, which also shows an unexpected cyclase activity. Our results pave the way for mining of hidden cyclases in biosynthetic pathways. They also open interesting prospects for the generation of novel biocatalysts for chemoenzymatic synthesis and pyran‐polyketides by combinatorial biosynthesis.     

Identification of novel sesterterpenes by genome mining of phytopathogenic fungi Phoma and Colletotrichum sp.

Two homologous gene clusters for the biosynthesis of sesterterpenes betaestacins were identified from two phytopathogens, Phoma betae and Colletotrichum orbiculare. Heterologous expression of identified oxidation enzymes with previously-characterized PbTS1 (BtcA_Pb) resulted in the production of seven novel sesterterpenes. Although both strains possessed homologous enzymes, oxidation state of corresponding products were different from each other, suggesting that structural diversification of sesterterpene skeletons might be achieved by these homologous enzymes with different functions.     

Aromatic polyketides from a caterpillar associated Alternaria sp.

Five new aromatic polyketides, alternaphenols A–E (1–5), and two known compounds (6–7) were isolated from a liquid culture of Alternaria sp. (strain no. NF2198), a caterpillar associated fungus. Their structures were determined by extensive spectroscopic analysis and single crystal X-ray crystallography. Compounds 1–7 were evaluated for their cytotoxic activities against a human skin melanoma A-375 cell line. Only compounds 4 and 5 showed weak cytotoxic activities.     

几丁质合成酶抑制剂

几丁质合成酶是生物合成几丁质的关键物质。几丁质是昆虫表皮和真菌细胞壁的特征成分,由于存在的特殊性而成为农药、医药研发的独特靶标。几丁质合成酶抑制剂由于具有安全、高效等特点,成为农用杀虫、杀螨、杀菌剂以及医药抗真菌药物的研发热点。本文综述了天然及人工合成的几丁质合成酶抑制剂的研究进展,并对其发展趋势进行了展望。     

Proton gradient energy in the catalytic ATP synthesis

ATP synthesis assisted by the F₁F₀-ATP synthase enzyme is considered in the context of chemical thermodynamics. It is shown that the proton gradient itself has no energy. For this reason, ATP synthesis with the participation of protons’ gradient has no privileges in comparison with direct ATP synthesis.     

肿瘤抑制因子WT1对人的诱导型一氧化氮合成酶基因的转录调节作用

将人的诱导型一氧化氮合成酶(hiNOS)启动子构建在带荧光素酶基因的载体pGL3-basic上, 构建成用荧光素酶为系统的启动子, 以研究载体p8.3iNOS. 结果显示, 肾母细胞肿瘤抑制因子(WT1)能够有效地抑制hiNOS启动子的转录; 且WT1的4个选择性剪接本的抑制效果有所不同, 其中WT1(-/-)在两种肝癌细胞(HepG2和Hep3B)中对hiNOS的表达均具有最强的抑制作用, 并且抑制效果具有剂量依赖性, 用Western blot检测结果进一步证实HepG2细胞中WT1(-/-)过量表达能下调hiNOS表达. 以上结果说明WT1在肝癌细胞中对人的hiNOS具有转录调节作用.     

两类脂肪酸合酶中酮酰还原酶的结构、功能和药物开发前景

自然界中脂肪酸合酶（FAS）以FASⅠ和FASⅡ两种形式存在。存在于动物中的FASⅠ是由多活性中心构成的复合酶,存在于植物和细菌中的FASⅡ则是由多个独立蛋白构成的酶体系。FASⅠ和FASⅡ中催化酮酰基团还原的酶有一定同源性,酶催化动力学和酶抑制动力学的性质相似,因分子量过大,目前尚无详细的FASⅠ空间结构信息。通过比较两种类型FAS中酮酰还原酶结构与功能的异同,进一步了解FASⅠ的结构与功能。对两种不同进化程度和存在形式的同源酶进行比较研究,将有得提示复合酶的结构与功能,并得寻找新型特异性蛋白抑制剂,为新药开发提供先导化合物,具有十分重要的理论意义和应用价值。     

注射酮色林对脊神经结扎大鼠脊髓背角和背根神经节nNOS表达的影响

通过结扎大鼠L5脊神经建立神经病理性痛模型.手术后第3日开始皮下注射酮色林(0.3 mg·kg-1)或其溶剂DMSO(体积分数为0.8%),每d1次,2周后灌流、取材,免疫组织化学实验显示神经结扎后L4背根神经节(DRG)和脊髓背角Ⅰ-Ⅱ层nNOS的表达均上调.然而皮下注射酮色林阻断5-HT2A受体,使L4 DRG和脊...