氢化酶重组表达研究进展

氢化酶催化最简单的氧化还原反应,但蛋白结构却非常复杂,对其蛋白结构和催化功能的研究牵动着生物制氢、光电产氢催化剂及氢能源电池等相关绿色能源产业的发展。氢化酶通常可逆地催化质子还原产氢的反应,对氧化还原电位非常敏感,催化活性中心易于被氧化失活,活性蛋白的分离提纯十分不易,使得对其催化机制的认识推进缓慢。为了获取更多的氢化酶活性蛋白,许多研究团队先后对氢化酶开展了大量的同源或异源重组表达研究,就这类研究工作进行了扼要的总结和分析。     

绿藻[FeFe]氢化酶

该文介绍了绿藻[FeFe]氢化酶的研究现状,包括酶的结构、催化中心、金属簇的性质,以及对氧的敏感性和可能的解决办法。并且对已报道的绿藻[FeFe]氢化酶基因及其调控等问题作了介绍。     

漆酶结构的研究进展

漆酶是一种含铜的多酚氧化酶,具有特异的氧化还原电位,能够催化氧化酚类和芳胺类化合物,同时伴随4个电子的传递,最终将O2还原成水。本文就近年来漆酶的结构及其特性的研究进展做扼要综述。     

普通脱硫弧菌D-2氢化酶的提纯与性质

从全国土壤腐蚀网站长辛店分离纯化到－株氢化酶活性较高的菌株D-2。经鉴定为普通脱硫弧菌(Desulfovibrio vulgaris)。该菌氢化酶主要位于菌体的周质空间。 用pH9．0的0．1mmol／L Tris—EDTA缓冲液将酶洗脱,经硫酸铵沉淀、DEAE-纤维素和sepbadex G-150柱层析纯化,酶活力达到205μmolH2·min-1·mg蛋白-1,得率为17．6％,纯化33．1倍。经SDS—PAGE和梯度-PAGE测定,该酶为一条肽链,分子量49 000道尔顿。该酶吸收光谱具有铁硫蛋白特征．Ε400nm和8 280Nm分别为34．8mM-1·cm-1和120mM-1·cm-1,每个酶分子含有12个铁原子和11个硫原子。N-溴代丁二酰胺完全抑制酶活力,Hg2+件的抑制率也达54％,但该酶对巯基封闭性试剂具有抗性。     

高等植物氢化酶活性研究进展

氢化酶作为一种可催化氢气氧化与质子还原的金属酶,在生物体的氢代谢过程中发挥着关键作用。已有研究表明,氢气干预可对植物的生长发育和抗逆性产生积极影响,同时一些高等植物的内源性产氢现象也已得到证实,然而关于催化内源性产氢的氢化酶目前了解较少。虽然已有多项研究表明,叶绿体可能是高等植物产氢的关键部位,但是鉴于多种植物在种子萌发时仍然可以产氢,而种子萌发过程中叶绿体还没有生成,加上氢化酶在进化上与线粒体复合物Ⅰ具有同源性,在对氢化酶研究现状进行概述的基础上,提出了高等植物线粒体具有氢化酶活性的猜想,并总结了线粒体存在氢化酶活性的初步实验证据,以期为后续线粒体与氢化酶的关系研究提供参考依据。     

普通脱硫弧菌D-2氢化酶的提纯与性质

从全国土壤腐蚀网站长辛店分离纯化到－株氢化酶活性较高的菌株D-2。经鉴定为普通脱硫弧菌(Desulfovibrio vulgaris)。该菌氢化酶主要位于菌体的周质空间。 用pH9．0的0．1mmol／L Tris—EDTA缓冲液将酶洗脱,经硫酸铵沉淀、DEAE-纤维素和sepbadex G-150柱层析纯化,酶活力达到205μmolH2·min-1·mg蛋白-1,得率为17．6％,纯化33．1倍。经SDS—PAGE和梯度-PAGE测定,该酶为一条肽链,分子量49 000道尔顿。该酶吸收光谱具有铁硫蛋白特征．Ε400nm和8 280Nm分别为34．8mM-1·cm-1和120mM-1·cm-1,每个酶分子含有12个铁原子和11个硫原子。N-溴代丁二酰胺完全抑制酶活力,Hg2+件的抑制率也达54％,但该酶对巯基封闭性试剂具有抗性。     

血脂异常相关酶的研究进展

血脂异常在临床上最常见的是高脂蛋白血症。随着经济的发展和人民生活水平的提高,高脂血症作为心脑血管疾病的重要危险因素,更加威胁着人类健康,对脂质异常和相关酶的研究也在逐渐深入。本文综述了几种与脂质代谢异常相关的关键酶及其研究进展。     

果胶甲酯酶的结构与功能研究进展

果胶甲酯酶(PME)是一种重要的果胶酶,其水解果胶中的甲酯基从而释放甲醇并降低果胶的甲酯化程度。目前在食品加工、茶饮料、造纸等生产工艺中有着广泛的应用前景。随着对PME的深入研究,已报道了几种不同来源的酶晶体结构,对这些已获得的晶体结构进行分析发现,PME属于右手平行β-螺旋结构,其催化残基为2个保守的天冬氨酸和1个谷氨酰胺残基,并且在催化过程中分别起到了一般酸碱、亲核试剂以及稳定中间体的作用。同时对其底物特异性进行分析,初步了解其底物与活性位点的识别机制。文中针对这几个相关方面进行了系统的综述。     

卡拉胶酶研究进展

卡拉胶酶是一种多糖水解酶,可以通过降解卡拉胶的β-1,4糖苷键,来生成卡拉胶低聚糖。根据酶解底物的差异,将其分为κ-卡拉胶酶,■-卡拉胶酶和λ-卡拉胶酶。近年来,研究发现卡拉胶寡糖具有抗病毒、抗肿瘤、免疫调节及抗凝血等药理活性,有望成为新一代的海洋药物而卡拉胶酶不但可以作为工具酶用于卡拉胶寡糖的制备,而且有助于卡拉胶结构的研究;另外,卡拉胶酶还可用于藻类原生质体的制备、因此,卡拉胶酶的研究具有重要的理论意义和明确的应用前景。本文从卡拉胶酶的分类及家族归属、来源、性质、分子生物学研究以及应用等方面综述了近年来国内外卡拉胶酶的最新研究进展     

色氨酸卤化酶研究进展

能催化卤代反应的卤化酶,因其具有高效、选择性好、反应条件温和的特点受到广泛关注。其中色氨酸卤化酶是研究最多的一类酶,它的特点是可以有选择性地卤化色氨酸,主要包括氯化和溴化。而卤代化合物具有多种生物活性,在医药、化工等领域有着广泛的应用。本文主要介绍了色氨酸卤化酶的来源和种类,酶学性质及其异源表达的研究现状;重点阐述了其结构和功能之间的相互关系及催化机理;并对色氨酸卤化酶的应用以及未来发展方向进行了展望,以期为色氨酸卤化酶的开发应用提供参考。     

Hydrogenase of purple bacteria: properties and regulation of synthesis

Physico-chemical properties of homogeneous preparations of soluble and membrane-bound hydrogenases from the purple sulfur bacterium Thiocapsa roseopersicina BBS and membrane-bound hydrogenase of Rhodopseudomonas capsulata, strain B10 have been studied. Compared to the enzymes from other sources, the hydrogenase of Thiocapsa roseopersicina is more stable to O₂ and products of its reduction (O ₂ ^- , H₂O₂), temperature and a number of other factors of the medium. A natural electron donor for T. roseopersicina hydrogenase is a low-potential cytochrome C₃, while the natural electron acceptors for hydrogenases of R. capsulata, T. roseopersicina, Ectothiorhodospira shaposhnikovii and Anabaena cylindrica are cytochromes of groups c and b.In different phototrophs, synthesis of hydrogenase can be inhibited by the presence of high concentrations of O₂. In some microorganisms (e.g. Rhodopseudomonas capsulata, strain B10) the repressing effect on hydrogenase formation is also exhibited by organic compounds. H₂ may not necessarily be present for hydrogenase synthesis by purple bacteria, but its presence may considerable increase the level of the enzyme.Abbreviations SDS sodium dodecylsulfate - Hipip high-potential iron — sulfur protein - R Rhodopseudomonas - T Thiocapsa - Rh Rhodospirillum - C Chromatium This paper is dedicated to Professor Dr. H.G. Schlegel in honour of his sixtieth birthday and in recognition of his great contribution in the field of physiology and biochemistry of microorganisms capable of using H₂. Professor H.G. Schlegel had a profound and most fuitful influence on the progress in the research of the laboratory headed by the author     

Hydrogenase from Acetobacterium woodii

Hydrogenase from fructose-grown cells of Acetobacterium woodii has been purified 70-fold to a specific activity of 3,500 mol hydrogen oxidized per min per mg of protein measured at 35°C and pH 7.6 with methyl viologen as electron acceptor. At the same conditions with reduced methyl viologen as electron donor the enzyme catalyzes the evolvement of 440 mol of H₂ per min per mg of protein. The enzyme was found in the soluble portion of the cell, indicating that it is either not membrane-bound or is loosely associated with the membrane. The purified enzyme, which does not contain nickel, exhibits spectroscopic properties similar to the iron-sulfur hydrogenase of Clostridium pasteurianum. The enzyme is strongly inhibited by carbon monoxide, with 50% inhibition occurring at approximately 7 nM CO. Ferredoxin, flavodoxin, and carbon monoxide dehydrogenase are reduced in hydrogen-dependent reaction by the A. woodi hydrogenase.Abbreviations CO dehydrogenase carbon monoxide dehydrogenase - MV methyl viologen - SDS sodium dodecyl sulfate This paper is dedicated to Professor Dr. Hans G. Schlegel on the occasion of his 60-years birthday. Hans' contributions to the field of microbiology are many and it is a pleasure for us to commemorate him in this way. One of us, L. G. L., had the fortune as a Humboldt-Preis recipient to spend a year at the Institut für Mikrobiologie der Universität Göttingen. Besides the best possible working conditions memories involve pleasant evenings with a glass of Rhine-wine in the Schlegels' home to strenuous back-packing in the Austrian Alps.     

Studies on the Induction of Reversible Hydrogenase from Cyanobacterium Anabaena variabilis and its Properties

The reversible hydrogenase in vegetative cells of A. variabilis cultured on NH4+ or N-free medium was induced by sparging with N2 for 24 hours under light. Both anaerobic condition and illumination appear to be necessary for the induction of hydrogenase in this algae. The properties of the hydrogenase in cell-free extract obtained from the cells grown on two nitrogen sources are similar: (1) Both the enzymes are able to evolve H2 in the presence of reduced methyl viotogen as electron donor, and to uptake H2 in the presence of benzyl viologen as electron acceptor. (2) The enzymes posses the thermal stability and are stable to O2. (3) The optimum pH required for H2 evolution activity of the enzymes is 7.0–7 5. (4) The Km of the enzymes obtained from NH4+ grown cells and N-free grown cells is 300 mmol/l and 295 mmol/l, respectively. So the high Km measured here suggests that the enzymes in both cases function physiologically as H2 evolution. (5) The activities of both enzymes are inhibited by CO but are not affected by C2H2. The induced H2 evolution activity of the reversible hydrogenase in cells grown on NH4+ reached 1530 nmol H2/mg dry wt, h, which was 3 to 5 times higher than from cells grown on N-free medium. Our experiment results indicate that the appearance of heterocysts of A. variabilis cultured on N-free medium affects the synthesis of reversible hydrogenase and the regulation of its activity.     

Structural Basis for the Reaction Mechanism of S-Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases

As a remarkable structural feature of hydrogenase active sites, [NiFe]-hydrogenases harbor one carbonyl and two cyano ligands, where HypE and HypF are involved in the biosynthesis of the nitrile group as a precursor of the cyano groups. HypF catalyzes S-carbamoylation of the C-terminal cysteine of HypE via three steps using carbamoylphosphate and ATP, producing two unstable intermediates: carbamate and carbamoyladenylate. Although the crystal structures of intact HypE homodimers and partial HypF have been reported, it remains unclear how the consecutive reactions occur without the loss of unstable intermediates during the proposed reaction scheme. Here we report the crystal structures of full-length HypF both alone and in complex with HypE at resolutions of 2.0 and 2.6 Å, respectively. Three catalytic sites of the structures of the HypF nucleotide- and phosphate-bound forms have been identified, with each site connected via channels inside the protein. This finding suggests that the first two consecutive reactions occur without the release of carbamate or carbamoyladenylate from the enzyme. The structure of HypF in complex with HypE revealed that HypF can associate with HypE without disturbing its homodimeric interaction and that the binding manner allows the C-terminal Cys-351 of HypE to access the S-carbamoylation active site in HypF, suggesting that the third step can also proceed without the release of carbamoyladenylate. A comparison of the structure of HypF with the recently reported structures of O-carbamoyltransferase revealed different reaction mechanisms for carbamoyladenylate synthesis and a similar reaction mechanism for carbamoyltransfer to produce the carbamoyl-HypE molecule.     

深海热液区化能自养菌Caminibacter profundus氢酶及其对H2的响应特点

化能自养菌中的氢酶在深海热液区生态系统的物质和能量转化中具有重要作用。以Caminibacter profundus为研究对象,通过设计PCR引物,克隆编码膜结合的类型I NiFe-氢酶大亚基基因序列hynL并进行生物信息学分析;研究hynL相对表达、甲基紫晶(MV)还原氢酶活性以及菌株生长对H2浓度变化的响应特点。结果表明,从C.profundus克隆获得864 bp的hynL基因片段,其编码的氨基端序列与Lebetimonas acidiphila的相似性为99%,与热液区化能自养的Epsilonproteobacteria D类群的类型I NiFe氢酶大亚基属同一分支。hynL相对表达量和MV还原的氢酶活性分别于12 h和24 h达到最高,此时菌体处于指数生长期;hynL相对表达量和MV还原的氢酶活性和菌株生长的最适H2浓度均为60%。提示C.profundus通过调控hynL的表达,响应环境中H2浓度的变化,以影响菌株能量代谢的催化过程和生长繁殖。     

台湾相思结瘤固氮与吸氢酶活性研究

台湾相思的根系具有多年生的根瘤,根瘤初发生时球状,以后发育成分叉瘤和扇状瘤。根瘤固氮活性因苗龄、成熟度不同而有明显差异。环境条件影响结瘤及固氮活性。15℃时结瘤受到明显抑制,固氮作用最适温度条件是25～30℃。光照不足降低根瘤固氮活性。短期轻度干旱不影响根瘤固氮活性,但持续干旱使固氮活性明显下降。pH4.5～8.5条件能正常结瘤,pH5.5时结瘤最好。根瘤固氮作用时不释放H_2,具有较高的吸氢酶活性,在固氮反应系统中加入5％的H_2,能提高根瘤固氮活性。     

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association,Assembly, Maturation,and Function

Hydrogenases are metalloenzymes that catalyze 2H⁺ + 2e⁻ ↔ H₂. A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)⁺ as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.     

A Bacterial Electron-bifurcating Hydrogenase

The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.     

Restoration of Escherichia coli hyd B Mutant Hydrogenase by Cultivation in Media Containing Various Metals

Hydrogenase restoration of Escherichia coli hydrogenase deficient mutant HK7, which carries a mutation at hyd B locus, was studied. Anaerobic growth of HK7 in the presence of iron chloride or vanadium chloride resulted in the restoration of hydrogen uptake activity of hydrogenase, but not hydrogen evolution activity. The growth of HK7 in the presence of nickel chloride restored total hydrogenase activity (hydrogen uptake and evolution) as Waugh and Boxer (1986) reported. Therefore, the leniency of HK7 hyd B product might permit the transportation and incorporation of iron chloride or vanadium chloride in hydrogenase, resulting in the alteration of hydrogenase activity.