Mutational Analysis of the Multicopy hao Gene Coding for Hydroxylamine Oxidoreductase in Nitrosomonas sp. Strain ENI-11

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao ₁, hao ₂, and hao ₃) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao ₁::kan, hao ₂::kan, or hao ₃::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao ₁::kan and hao ₃::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.     

Characterization of the reaction of decoupling ubiquinone with bovine mitochondrial respiratory complex I

We previously produced the unique ubiquinone QT (“decoupling” quinone), the catalytic reduction of which in NADH-quinone oxidoreduction with bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) is completely decoupled from proton translocation across the membrane domain. This feature is markedly distinct from those of typical short-chain quinones such as ubiquinone-1. To further characterize the features of the QT reaction with complex I, we herein synthesized three QT analogs, QT2–QT4, and characterized their electron transfer reactions. We found that all aspects of electron transfer (e.g. electron-accepting activity and membrane potential formation) vary significantly among these analogs. The features of QT2 as decoupling quinone were slightly superior to those of original QT. Based on these results, we conclude that the bound positions of QTs within the quinone binding cavity susceptibly change depending on their side-chain structures, and the positions, in turn, govern the behavior of QTs as electron acceptors.     

Steric hindrance controls pyridine nucleotide specificity of a flavin‐dependent NADH:quinone oxidoreductase

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD⁺ to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD⁺ binds in a folded conformation at the interface of the TIM‐barrel domain and the extended domain of the enzyme. Comparison of the enzyme‐NAD⁺ structure with that of the ligand‐free enzyme revealed a different conformation of a short loop (75–86) that is part of the NAD⁺‐binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD⁺ within the binding pocket. An interrupted helix consisting of two α‐helices connected by a small three‐residue loop binds the pyrophosphate moiety of NAD⁺. The adenine moiety of NAD⁺ appears to π–π stack with Y261. Steric constraints between the adenosine ribose of NAD⁺, P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.     

心理应激对小鼠脂肪组织黄嘌呤氧化酶表达、活性及相关指标的影响*

目的:探讨心理应激对小鼠脂肪组织黄嘌呤氧化酶表达、活性及相关指标的作用。方法:雄性无特定病原体(SPF)级20只昆明小鼠随机分2组(每组10只),即慢性束缚应激(Stress)组和正常对照(Control)组。Stress组小鼠每天在自制式束缚器中限制活动2 h,其余时间两组小鼠在相同环境中自由饮水摄食,实验持续14 d,取血和白色脂肪组织(WAT);观察脂肪组织病理学改变,检测WAT中黄嘌呤氧化酶(XO)和烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(Nox-4)的蛋白水平,检测WAT组织中XO、Nox-4、超氧化物歧化酶(Mn SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、脂联素(ADPN)、单核细胞趋化蛋白1(MCP-1)、白介素6(IL-6)、肿瘤坏死因子α(TNF-α)、胰岛素受体底物1(IRS-1)、葡萄糖转运蛋白4(GLUT-4)、组织因子(TF)、纤溶酶原激活物抑制物1(PAI-1)的mRNA表达,检测血清和WAT组织中XO酶活性以及血清甘油三酯(TG)、总胆固醇(T-Cho)、游离脂肪酸(FFA)、尿酸(UA)的含量。结果:与control组比较,stress小鼠腹股沟WAT组织中XO免疫染色阳性着色细胞黄褐色沉淀深且丰富,WAT中出现大量的单核细胞、中性粒细胞、嗜酸性粒细胞及浆细胞浸润反应和炎症性的改变;血清XO浓度、WAT组织中XO mRNA水平和XO的酶活性显著升高(P＜0.01),血清游离脂肪酸(FFA)和尿酸(UA)的含量显著增高(P＜0.01),WAT组织中Nox-4蛋白、MCP-1、IL-6、TNF-α、TF、PAI-1mRNA的表达水平显著增高(P＜0.01),而Mn-SOD、GSH-Px、CAT、ADPN、IRS-1和GLUT-4的mRNA水平则显著降低(P＜0.01)。结论:心理应激可诱发脂肪XO过量表达及其活性增高,进而引起脂肪炎症、糖代谢及凝血酶原异常等反应。     

番茄总皂苷对小鼠高尿酸血症的调节作用北大核心CSCD

为研究番茄总皂苷对尿酸的调节作用,该文以番茄水提物为试材,利用次黄嘌呤和氧嗪酸钾以及尿酸和氧嗪酸钾建立高尿酸模型小鼠,考察番茄总皂苷对正常小鼠及高尿酸血症小鼠尿酸排泄量、血尿酸、尿素氮、肌酐、黄嘌呤氧化酶以及脏器指数的影响。结果表明:番茄总皂苷不影响正常小鼠血尿酸水平,正常组及番茄低、中、高剂量组血尿酸值分别为(170.4±36.7)、(178.3±69.7)、(175.5±42.1)、(185.3±72.6)μmol·L^(-1);番茄总皂苷对次黄嘌呤和氧嗪酸钾联合诱导的高尿酸血症小鼠可以降低血尿酸水平,降低黄嘌呤氧化酶活性,正常组、模型组及番茄高剂量组血尿酸值分别为(140.4±36.7)、(378.3±69.7)、(278.3±62.6)μmol·L^(-1),正常组、模型组及番茄低、中、高剂量组黄嘌呤氧化酶值分别为(1.2±0.3)、(1.8±0.2)、(1.6±0.2)、(1.5±0.3)、(1.3±0.4)U·g^(-1) liver;对尿酸和氧嗪酸钾联合诱导的高尿酸血症小鼠,可降低血尿酸水平,降低黄嘌呤氧化酶活性,正常组、模型组及番茄高剂量组血尿酸值分别为(98.8±21.8)、(455.6±78.8)、(333.7±68.7)μmol·L^(-1),正常组、模型组及番茄高剂量组黄嘌呤氧化酶值分别为(2.1±0.3)、(2.5±0.2)、(2.3±0.2)U·g^(-1) liver。综上结果表明,番茄总皂苷不影响正常小鼠血尿酸水平,但能降低高尿酸模型小鼠的血尿酸水平,其机制可能与降低黄嘌呤氧化酶活性有关。     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Integration of the Mitochondrial-Processing Peptidase into the Cytochrome bc 1 Complex in Plants

The plant mitochondrial cytochrome bc ₁ complex, like nonplant mitochondrial complexes,consists of cytochromes b and c ₁, the Rieske iron–sulfur protein, two Core proteins, and fivelow-molecular mass subunits. However, in contrast to nonplant sources, the two Core proteinsare identical to subunits of the general mitochondrial processing peptidase (MPP). The MPPis a fascinating enzyme that catalyzes the specific cleavage of the diverse presequence peptidesfrom hundreds of the nuclear-encoded mitochondrial precursor proteins that are synthesizedin the cytosol and imported into the mitochondrion. Integration of the MPP into the bc ₁complex renders the bc ₁ complex in plants bifunctional, being involved both in electrontransport and in protein processing. Despite the integration of MPP into the bc ₁ complex,electron transfer as well as translocation of the precursor through the import channel areindependent of the protein-processing activity. Recognition of the processing site by MPPoccurs via the recognition of higher-order structural elements in combination with charge andcleavage-site properties. Elucidation of the three-dimensional (3-D) structure of the mammaliancytochrome bc ₁ complex is highly useful for understanding of the mechanism of action of MPP.In memory of my teacher—an insightful, devoted, and enthusiastic scientist and an amiable and kind-hearted human being—Lars Ernster     

Structure of the Avian Mitochondrial Cytochrome bc 1 Complex

There are now four structures of vertebrate mitochondrial bc ₁ complexes available in theprotein databases and structures from yeast and bacterial sources are expected soon. Thisreview summarizes the new information with emphasis on the avian cytochrome bc ₁ complex(PDB entries 1BCC and 3BCC). The Rieske iron–sulfur protein is mobile and this has beenproposed to be important for catalysis. The binding sites for quinone have been located basedon structures containing inhibitors and, in the case of the quinone reduction site Qi, thequinone itself.     

Protochlorophyllide-NADP+ and protochlorophyllide- NADPH complexes and their regeneration after flash illumination in leaves and etioplast membranes of dark-grown wheat

The fast (1 min) regeneration process of the photoactive Pchlide forms after a light flash was studied in etiolated wheat leaves, and this process was simulated in vitro by incubating etioplast inner membranes of wheat with excess NADPH or NADP+. The 77 K fluorescence spectra were recorded after flash illumination, dark incubation and a subsequent flash illumination of the samples. A non-photoactive Pchlide form with an emission maximum at 650 nm was transiently detected in leaves during regeneration of a photoactive Pchlide form with an emission maximum at 654 nm. Gaussian deconvolution of fluorescence spectra of isolated membranes showed that this 650 nm form appeared in conditions of excess NADP+, as suggested in previous studies. Additionally a Pchlide form emitting at 638.5 nm was detected in the same conditions. The analysis of the spectra of leaves at different times after a flash indicated that these two non-photoactive forms are involved as intermediates in the regeneration of photoactive Pchlide. This regeneration is in correlation with the production of the Chlide form emitting at 676 nm. The results demonstrate that, in vivo, part of the NADPH:protochlorophyllide oxidoreductase is reloading with nonphotoactive Pchlide on a fast time-scale and that the 676 nm Chlide form is the released product of the phototransformation in this process.     

The importance of<Emphasis Type="Italic"> porE</Emphasis> and<Emphasis Type="Italic"> porF</Emphasis> in the anabolic pyruvate oxidoreductase of<Emphasis Type="Italic"> Methanococcus maripaludi</Emphasis>s

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase - POR Pyruvate oxidoreductase