土壤水解酶类催化动力学研究进展

土壤水解酶是存在于土壤中的一种重要的酶类,参与了土壤中为数众多的重要生物化学反应,与土壤中多种营养元素转化密切相关其催化反应的动力学研究常用来阐明其催化过程的特性、酶的本质属性及其对环境变化的响应等,研究土壤水解酶动力学特征对探讨其来源、性质及影响因素,对进一步调控多种营养元素参与的反应过程有着重要意义。文中概述了土壤水解酶的种类及其参与的生物化学反应;探讨了土壤水解酶动力学研究的理论基础;综述了土壤水解酶催化动力学研究的进展和影响因素,在此基础上,对今后研究提出了几点建议。     

细菌氧化还原酶烟酰型辅酶偏好性的分子机制

氧化还原酶因其在催化制备手性醇、羟基酸、氨基酸等方面的极大优势,在精细化工、农药、制药、食品等领域具有重要的用途。大多数氧化还原酶需要在烟酰胺类辅酶的参与下才能完成催化反应,且表现出对此类辅酶使用的偏好性。本文基于对细菌氧化还原酶和烟酰型辅酶的结构分析,总结了代表性的氧化还原酶的辅酶结合部位的结构特征以及决定辅酶偏好性的氨基酸性质,为揭示氧化还原酶对烟酰型辅酶利用偏好性的分子机制和实现辅酶偏好性改造提供依据。     

植物体内一氧化氮合成途径研究进展

一氧化氮(NO)作为一种气体信号分子,在植物生理过程中发挥重要作用,它参与调节植物的生长、发育及对外界环境的应激反应.植物体内主要通过酶催化途径和非酶催化途径合成NO.酶催化途径合成NO的主要酶包括一氧化氮合酶(nitric oxide synthase,NOS)和硝酸还原酶(nitrate reductase,NR),以及在某些植物的特定组织或器官或在特殊环境条件下存在的一氧化氮氧化还原酶(nitric oxide oxidoreductase,Ni-NOR)和黄嘌呤氧化还原酶(xanthine oxidoreductase,XOR).非酶催化合成途径主要是在酸性和还原剂存在条件下将亚硝酸盐还原成NO.该文主要结合研究方法,综述了植物体内NO合成途径的研究进展,为植物体内NO信号的作用机理的深入研究提供信息资料.     

高通量筛选具有催化活性和立体选择性羰基还原酶

根据羰基还原酶催化可逆氧化还原反应的原理,利用与偶氮还原酶催化偶氮染料还原反应耦合的颜色变化,建立了一种新的羰基还原酶筛选方法。由于羰基还原酶在催化醇底物氧化反应时会产生NAD(P)H,当在反应体系中加入偶氮还原酶AzoB和偶氮染料金橙Ⅰ的时候,偶氮还原酶可以利用NAD(P)H作为电子的供体与底物金橙Ⅰ发生反应,导致反应体系颜色的变化,这样就能够根据明显的颜色变化推断出该羰基还原酶是否对所选底物表现出特定的活性,进而可以筛选出有活性的羰基还原酶。同时,使用不同构型的手性醇作为底物时,根据体系的颜色变化,可以实现羰基还原酶的活性和立体选择性的同时筛选。     

细胞色素P450酶催化反应动力学研究进展

细胞色素P450是内质网膜上混合功能氧化酶系统的末端氧化酶,在生物体内分布广泛,主要催化机体内源和外源性物质在体内的氧化反应.细胞色素P450种类的多样性、催化反应类型的多样性以及底物的广谱性使其成为自然界最具催化作用的生物催化剂.在临床药物的生物学转化中,它参与大部分药物的生物氧化,因此具有重要的生物学意义.本文主要对细胞色素P450的催化反应机理,尤其是细胞色素P45催化下乙醇氧化的反应机理,及其在药代动力学方面的研究进行了综述.     

正交氧化还原体系及其应用

氧化还原反应是最常见的代谢反应类型之一,其中绝大部分通过辅因子依赖型氧化还原酶催化实现.由于辅因子广泛参与细胞内氧化还原反应及其他生物学过程,因代谢途径改造而扰动辅因子水平的生物学效应尚难以预测.设计构建基于人工辅因子的正交体系,是减少人工代谢途径与内源代谢网络相互干扰、降低系统复杂度、提高调控代谢网络有效性的新策略.本文探讨了正交氧化还原体系的构建方法,并结合实例说明其对提高能量传递特异性和人工代谢途径效率的重要意义.     

生物催化立体选择性氧化还原中存在问题及其发展策略

以立体选择性氧化还原酶或其全细胞催化的不对称氧化还原反应已经成为转化光学活性手性醇及其他手性化合物的有效手段。然而,生物催化氧化还原反应体系存在着催化活性与专一性、反应体系与催化稳定性等生物催化剂所固有的局限性问题,而且,生物氧化还原反应必需辅酶及其再生问题也是限制该转化途径产业化应用的一个重要因素。围绕上述生物催化立体选择性氧化还原中存在的关键问题,现代分子生物技术及反应工程的不断突破和发展为改善生物催化立体选择性氧化还原在催化剂本身和反应工程方面的局限性提供了有效的发展策略,为其进一步大规模产业应用提供了发展基础。     

烟曲霉FADB基因参与菌株耐药的潜在机理

FAD结合的氧化还原酶编码基因FADB(GenBank ID:Afu4g14630)的编码产物在烟曲霉中为一种与E4D结合的氧化还原酶,可能参与真菌的呼吸.为了探究其具体功能,本研究通过克隆烟曲霉E4DB基因,构建烟曲霉FADB基因的敲除株,了解该基因对烟曲霉药物敏感性、渗透压、氧化压力物质敏感性的作用机理.采用高通量...     

酵母醇脱氢酶在反向胶团中的某些特性

酵母醇脱氢酶在反向胶团中的某些特性张强,袁静明,赵葆华（山西大学分子科学研究所,太原０３０００６）反向胶团酶催化是酶学研究的新领域,已涉及到水解酶、转移酶、氧化还原酶等的研究~［１］。醇脱氢酶是糖代谢路线中重要的氧化还原酶之一,但不同来源的醇脱氢酶分?..     

贡嘎山海拔梯度上不同植被类型土壤甲烷氧化菌群落结构及多样性

甲烷是仅次于CO₂的第二大温室气体.森林表层土壤中甲烷好氧氧化作用是大气甲烷重要的汇,在碳循环和减缓全球变暖方面起着重要作用.研究不同植被类型土壤中甲烷氧化菌的群落结构及多样性,有助于更好地理解植被演替、人为干扰和不同土地利用背景下甲烷氧化菌群落组成和多样性变化与地上植被之间的相互关系.本研究在贡嘎山东坡海拔梯度上的4种不同植被类型中采集了92个土壤样品,利用Miseq测序技术和生物信息学方法评估了甲烷氧化菌群落组成及多样性在4种不同植被类型间的变化,并探讨了其变异的潜在原因.结果表明: 常绿阔叶林和针阔叶混交林土壤中甲烷氧化菌的群落结构较为相似,暗针叶林和灌丛草甸土壤甲烷氧化菌的群落结构较为相似.4种不同植被生态系统中,针阔叶混交林土壤中的甲烷氧化菌α多样性显著高于其他3种植被生态系统(P＜0.001),且暗针叶林和灌丛草甸土壤中甲烷氧化菌β多样性显著高于常绿阔叶林和针阔叶混交林(P＜0.001).Spearman相关分析表明,不同类型甲烷氧化菌的相对丰度对环境变化的响应模式不同.造成α多样性差异的主要因子可能是土壤总氮、电导率和土壤温度.偏Mantel检验分析和冗余分析(RDA)表明,常绿阔叶林和针阔叶混交林土壤甲烷氧化菌多样性受环境因子的影响较大,而暗针叶林和灌丛草甸土壤中甲烷氧化细菌多样性变化可能存在其他潜在的影响因素或者机制.降水可能是造成低海拔常绿阔叶林和针阔叶混交林与高海拔暗针叶林和灌丛草甸土壤甲烷氧化细菌群落结构差异的主要原因.贡嘎山海拔梯度上不同植被类型土壤中甲烷氧化菌的群落结构和多样性变化可能主要是由于土壤理化性质和气候变化综合作用的结果.     

Mitochondrial respirasome works as a single unit and the cross-talk between complexes I,III2 and IV stimulates NADH dehydrogenase activity

Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III₂, and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O₂ and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III₂ and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production.     

The O2- generating oxidoreductase of human neutrophils: evidence of an obligatory requirement for calcium and magnesium for expression of catalytic activity

NADPH-dependent O2- generating oxidoreductase activity recovered from cell lysates of phorbol myristate acetate-stimulated human neutrophils exhibits dependence on Ca+2 and Mg+2 for full expression of its catalytic activity. O2- generating activity was completely abolished by exposure of the oxidoreductase to EDTA, then reconstituted by exposure of the enzyme to Ca+2 and Mg+2 in excess of the EDTA concentration used to block catalytic activity. The oxidoreductase responded maximally to either 0.25 mM Ca+2 or 0.80 mM Mg+2. The pH optimum of the oxidoreductase exposed to Ca+2 and Mg+2 is between pH 7.0 and 7.6. The molar ratio of NADPH oxidation to O2- production determined at pH 7.6 in the presence of Ca+2 and Mg+2 is 0.49, indicating 1 mole of NADPH oxidized per 2 moles of O2- formed. Particulate fractions recovered from cell lysates of resting neutrophils exhibited no oxidoreductase activity under the same conditions.     

Kinetics of the oxidoreductase involved in the conversion of O-methylsterigmatocystin to aflatoxin B1

Among the enzymatic steps in the aflatoxin biosynthetic pathway, the conversion of O-methylsterigmatocystin (OMST) to the potent environmental carcinogen aflatoxin B1 (AFB1), has been proposed to be catalysed by an oxidoreductase (OR) that requires a cytochrome P-450 type of oxidoreductase activity. This enzyme displays relative specificity towards OMST homologues in fungal whole cells. These studies were extended to the action of a cell-free enzyme system (CFES), on five OMST homologues, with a view to establish the kinetics. In the current study a CFES, containing an oxidoreductase, was derived from a blocked mutant of Aspergillus parasiticus (Wh1-11-105). The key experimental steps involved rapid concentration and efficient dialysis by membrane filtration to remove small biomolecules (MW<10,000), co-factors, primary and secondary metabolites. The kinetic parameters of the enzyme-substrate reactions indicated that the reaction follows a Michealis-Menten kinetics and OR activity decreased in the order: O-butylsterigmatocystin>O-propylsterigmatocystin>O-ethylsterigmatocystin>O-methylsterigmatocystin>O-acetylsterigmatocystin>O-benzoylsterigmatocystin. The 7-O-alkyl homologues were the best substrate for the CFES, thereby substantially supporting that the 7-O-methyl group of OMST is preferred for OR catalytic activity in the absence of any other alkylating groups in vitro. The Km was calculated as 5.65 microM for this CFES and varied marginally among the OMST homologues studied.     

The pathway of electron transfer in the dimeric QH2: Cytochromec oxidoreductase

The experimental data currently available suggest that QH₂: cytochromec oxidoreductase functions according to a Q-cycle type of mechanism. The molecular weight of the enzyme in a natural or artificial phospholipid bilayer or in solution corresponds to that of a dimer. The pre-steady state kinetics of reduction of the prosthetic groups indicate that the enzyme is functionally dimeric. A double Q cycle is proposed, describing the pathway of electron transfer in the dimeric QH₂: cytochromec oxidoreductase. According to this scheme, the two monomeric halves of the enzyme act in a cooperative fashion to complete the catalytic cycle. It is proposed that high-potential cytochromeb-562 and low-potential cytochromeb-562 act cooperatively, viz. as a functional pair, in the antimycin-sensitive reduction of ubiquinone to ubiquinol.     

Insight into disulfide bond catalysis in Chlamydia from the structure and function of DsbH, a novel oxidoreductase

The Chlamydia family of human pathogens uses outer envelope proteins that are highly cross-linked by disulfide bonds but nevertheless keeps an unusually high number of unpaired cysteines in its secreted proteins. To gain insight into chlamydial disulfide bond catalysis, the structure, function, and substrate interaction of a novel periplasmic oxidoreductase, termed DsbH, were determined. The structure of DsbH, its redox potential of -269 mV, and its functional properties are similar to thioredoxin and the C-terminal domain of DsbD, i.e. characteristic of a disulfide reductase. As compared with these proteins, the two central residues of the DsbH catalytic motif (CMWC) shield the catalytic disulfide bond and are selectively perturbed by a peptide ligand. This shows that these oxidoreductase family characteristic residues are not only important in determining the redox potential of the catalytic disulfide bond but also in influencing substrate interactions. For DsbH, three functional roles are conceivable; that is, reducing intermolecular disulfides between proteins and small molecules, keeping a specific subset of exported proteins reduced, or maintaining the periplasm of Chlamydia in a generally reducing state.     

A conserved histidine-aspartate pair is required for exovinyl reduction of biliverdin by a cyanobacterial phycocyanobilin:ferredoxin oxidoreductase

Phycocyanobilin:ferredoxin oxidoreductase is a member of the ferredoxin-dependent bilin reductase family and catalyzes two vinyl reductions of biliverdin IXalpha to produce phycocyanobilin, the pigment precursor of both phytochrome and phycobiliprotein chromophores in cyanobacteria. Atypically for ferredoxin-dependent enzymes, phycocyanobilin:ferredoxin oxidoreductase mediates direct electron transfers from reduced ferredoxin to its tetrapyrrole substrate without metal ion or organic cofactors. We previously showed that bound bilin radical intermediates could be detected by low temperature electron paramagnetic resonance and absorption spectroscopies (Tu, S., Gunn, A., Toney, M. D., Britt, R. D., and Lagarias, J. C. (2004) J. Am. Chem. Soc. 126, 8682-8693). On the basis of these studies, a mechanism involving sequential electron-coupled proton transfers to protonated bilin substrates buried within the phycocyanobilin:ferredoxin oxidoreductase protein scaffold was proposed. The present investigation was undertaken to identify catalytic residues in phycocyanobilin:ferredoxin oxidoreductase from the cyanobacterium Nostoc sp. PCC7120 through site-specific chemical modification and mutagenesis of candidate proton-donating residues. These studies identified conserved histidine and aspartate residues essential for the catalytic activity of phycocyanobilin:ferredoxin oxidoreductase. Spectroscopic evidence for the formation of stable enzyme-bound biliverdin radicals for the H85Q and D102N mutants support their role as a "coupled" proton-donating pair during the reduction of the biliverdin exovinyl group.     

Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase

Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine.     

Steady-state kinetics and inhibitory action of antitubercular phenothiazines on mycobacterium tuberculosis type-II NADH-menaquinone oxidoreductase (NDH-2)

Type-II NADH-menaquinone oxidoreductase (NDH-2) is an essential respiratory enzyme of the pathogenic bacterium Mycobacterium tuberculosis (Mtb) that plays a pivotal role in its growth. In the present study, we expressed and purified highly active Mtb NDH-2 using a Mycobacterium smegmatis expression system, and the steady-state kinetics and inhibitory actions of phenothiazines were characterized. Purified NDH-2 contains a non-covalently bound flavin adenine dinucleotide cofactor and oxidizes NADH with quinones but does not react with either NADPH or oxygen. Ubiquinone-2 (Q2) and decylubiquinone showed high electron-accepting activity, and the steady-state kinetics and the NADH-Q2 oxidoreductase reaction were found to operate by a ping-pong reaction mechanism. Phenothiazine analogues, trifluoperazine, Compound 1, and Compound 2 inhibit the NADH-Q2 reductase activity with IC50 = 12, 11, and 13 microm, respectively. Trifluoperazine inhibition is non-competitive for NADH, whereas the inhibition kinetics is found to be uncompetitive in terms of Q2.     

Na<Superscript>+</Superscript>-translocating NADH: Quinone oxidoreductase: Progress achieved and prospects of investigations

Structural and catalytic properties of bacterial Na⁺-translocating NADH: quinone oxidoreductases are briefly described. Special attention is given to studies on kinetics of the enzyme interaction with NADH and the role of sodium ions in this process. Based on the existing data, possible model mechanisms of sodium transfer by Na⁺-translocating NADH:quinone oxidoreductase are proposed.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 177–185.Original Russian Text Copyright © 2005 by Bogachev,Verkhovsky.This revised version was published online in April 2005 with corrections to the post codes.