醌血红素蛋白的电子传递

醌蛋白是以吡咯喹啉醌（PQQ）及其结构类似物为辅酶的一类氧化还原酶。醌血红素蛋白是以PQQ和一个或多个血红素作为辅助因子的醌蛋白,包括醌血红素蛋白醇脱氢酶和醌血红素蛋白胺脱氢酶。简要综述了醌血红素蛋白的结构特点,在分子内从PQQ到血红素的电子传递,以及醌血红素蛋白与蛋白之间的分子间的电子传递。     

酮古龙酸菌与呼吸链偶联的2-KGA代谢途径研究进展

酮古龙酸菌可将底物L-山梨糖转化为维生素C的前体2-酮基-L-古龙酸(2-KGA)。该菌共存在5种反应参与2-KGA代谢,包括:1D-山梨醇氧化为L-山梨糖;2L-山梨糖氧化为L-山梨酮;3L-山梨酮(吡喃型)氧化为2-KGA;4L-山梨酮(呋喃型)氧化为维生素C。52-KGA还原为L-艾杜糖酸。其中L-山梨糖/L-山梨酮脱氢酶(SSDH)参与反应123,L-山梨糖脱氢酶(SDH)参与反应23,L-山梨酮脱氢酶(SNDH)参与反应34,醛脱氢酶(ALDH)参与反应3,2-KGA还原酶(2-KGR)参与反应5。SDH/SSDH/ALDH属于Ⅰ型醌酶,其辅酶为1分子PQQ;SNDH属Ⅱ型醌酶,与PQQ、heme C共同构成quinohemoproteins,2种醌酶均分布于周质空间中与呼吸链相偶联,意味着这种膜上直接氧化过程伴随ATP产生,使得菌体可以利用环境中的底物实现快速供能。     

吡咯喹啉醌生物合成研究进展

吡咯喹啉醌(PQQ)是一种较新近发现的氧化还原酶的辅酶,对微生物及动植物均具有重要生理作用。已知能产生PQQ的生物仅限于某些革兰阴性细菌,已分离得到几种不同来源的PQQ生物合成基因,其序列具有一定的保守性。PQQ的生物合成涉及4～7个基因,这些基因一般成簇排列。业已证明,谷氨酸和酪氨酸是PQQ合成的前体物质。对各个基因的功能已有不同程度的了解,但PQQ的生物合成途径还尚未阐明。     

吡咯喹啉醌(PQQ)营养作用研究进展

吡咯喹啉醌(PQQ)是细菌脱氢酶氧化还原反应的辅助因子,广泛存在于微生物、植物、动物及人体中。迄今为止,PQQ催化氧化还原反应的能力远超过已知的生物活性分子。体内外研究表明,PQQ能够刺激微生物生长,增强其对极端环境的适应能力,并对植物和动物的生长、发育和繁殖十分重要。本文阐述了PQQ的理化性质、自然分布和营养作用的研究进展,以推动其在食品、医疗及农林渔业领域的发展应用。     

利用山梨糖脱氢酶活性筛选氧化葡糖杆菌PQQ合成基因簇

【目的】利用山梨糖脱氢酶醌酶活性从氧化葡糖杆菌H24中分离PQQ生物合成基因簇。【方法】利用ptsG位点整合sdh基因的大肠杆菌JM109作为宿主菌构建了氧化葡糖杆菌H24的基因组DNA文库。通过山梨糖脱氢酶活性检测,从文库中筛选具有PQQ合成能力的单菌落并进行亚克隆。【结果】从氧化葡糖杆菌H24的基因组文库中筛选得到一株具有山梨糖脱氢酶活性的单菌落,亚克隆后序列分析显示插入片段全长5400bp,对应5个编码框(pqqABCDE),与其他细菌PQQ生物合成基因簇有很高的序列同源性。【结论】利用山梨糖脱氢酶醌酶活性成功从氧化葡糖杆菌H24中分离克隆得到了PQQ生物合成基因簇pqqABCDE。     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

小红参醌抑制烫伤小鼠肠道细菌易位

近年来,创伤后肠道细菌易位引起的内源性感染日益受到重视。为了探讨细菌易位的机制,本文采用具有一定的抗氧化作用并能减轻自由基损伤、改善PMN功能的小红参醌进行试验。试验分为两组,Ⅰ组26只Balb/C小鼠管饲小红参醌液(5mg/ml)0.5ml三天,Ⅱ组21只Balb/c小鼠管饲无菌蒸馏水0.5ml三天,而后行25％Ⅲ°烫伤。伤后24小时处死,观察细菌易位发生率,回肠MDA和SOD含量,及病理组织学改变。观察的结果为:Ⅰ组肠道细菌易位至MLN发生率为15.4％,Ⅱ组为57.14％,两者相比有显著性差异(P<0.01)。Ⅰ组MDA含量为7.25±1.74n mol/克蛋白,Ⅱ组为11.00±1.74n mol/克蛋白,两者有显著性差异(P<0.01),但SOD含量无统计学意义。病理形态观察支持了上述结果。提示小红参醌具有抑制伤后小鼠肠道细菌易位的作用。     

一株新的产吡咯喹啉醌甲基营养菌全基因组测序和比较基因组学分析

【背景】由于甲基营养菌被发现的时间较短,而且可以生产吡咯喹啉醌(pyrroloquinoline quinone,PQQ)的甲基杆菌属细菌只有少数菌株的全基因组序列被公布,增加了该类细菌基因组学和生物代谢途径研究的难度。【目的】将本实验室筛选的PQQ生产菌经多种诱变方式处理,用于提高PQQ的发酵产量。对高产突变菌株进行全基因组解析,以探究甲基杆菌PQQ合成的分子机制,为后续分子育种提供序列背景信息。【方法】将野生型PQQ生产菌株进行紫外诱变、亚硝基胍诱变、甲基磺酸乙酯诱变、硫酸二乙酯诱变和紫外-氯化锂复合诱变。将突变菌株利用PromethION三代测序平台和MGISEQ-2000二代测序平台测序,然后进行组装和功能注释。组装得到的全基因组序列与模式菌株扭脱甲基杆菌AM1 (Methylobacterium extorquens AM1)进行比较基因组学分析。【结果】经11轮诱变获得一株突变菌株NI91,其PQQ产量为19.49 mg/L,相较原始菌株提高44.91%。突变菌株NI91的基因组由一个5 409 262 bp的染色体组成,共编码4 957个蛋白,与模式菌株M. extorquens AM1比较发现其PQQ合成过程中剪切加工相关的基因pqqF和pqqG缺失,但首次在甲基营养菌中发现与基因pqqF具有相似功能的基因pqqL,且基因pqqC/D的序列存在较大差异。【结论】为甲基营养类细菌甲基杆菌的功能基因组学研究及PQQ合成机理研究提供了基础数据支持,NI91与模式菌株M. extorquens AM1的比较基因组学分析为揭示PQQ合成的不同机理提供了分子基础。     

常压室温等离子体诱变扭脱甲基杆菌AM1高产吡咯喹啉醌

吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)作为一种新型的氧化还原酶辅酶,在医药和食品等领域有广阔的应用前景。为改善扭脱甲基杆菌Methylobacterium extorquens AM1 PQQ生产性能,采用常压室温等离子体(Atmospheric and room temperature plasma,ARTP)进行诱变,结合高通量快速筛选方法,得到以PQQ产量为指标的正向突变株。ARTP诱变的菌株正突变率为31.6%,筛选得到的较优正突变株M.extorquens AM1(E-F3),PQQ产量达到54.0 mg/L,是出发菌株的近3倍。系统的高通量方法筛选ARTP诱变菌为后续进一步提高M.extorquens AM1菌株PQQ的产量奠定了基础,亦为改善菌株生产性能提供了新思路。     

吡咯喹啉醌产生菌筛选方法建立及菌种筛选

吡咯喹啉醌(PQQ)是一种氧化还原酶的辅酶,具有多种生理功能。扩增得到大肠杆菌葡萄糖脱氢酶(GDH)基因,并利用表达载体pET28a在E.coli BL21(DE3)中进行了表达。纯化了可溶性表达产物,并建立了基于GDH的重组酶法分析PQQ的方法。确定了甲基营养菌筛选模型,从2000余份土样中分离得到一株PQQ高产生菌MP606,在未经培养条件优化及诱变选育的条件下PQQ产量达113mg/L。从该菌培养液中制备得到了产物的结晶,HPLC分析、特征光谱分析以及酶法分析均证实该产物为PQQ。扩增并分析了MP606的16S rDNA序列,结果显示该菌16S rDNA序列与12种甲基营养菌都具有95%以上同源性,其中与食甲基菌属两菌株的16S rDNA序列同源性达99%。     

Functional prediction: identification of protein orthologs and paralogs

Orthologs typically retain the same function in the course of evolution. Using beta-decarboxylating dehydrogenase family as a model, we demonstrate that orthologs can be confidently identified. The strategy is based on our recent findings that substitutions of only a few amino acid residues in these enzymes are sufficient to exchange substrate and coenzyme specificities. Hence, the few major specificity determinants can serve as reliable markers for determining orthologous or paralogous relationships. The power of this approach has been demonstrated by correcting similarity-based functional misassignment and discovering new genes and related pathways, and should be broadly applicable to other enzyme families.     

Alcohol Dehydrogenases: Identification and Names for Gene Families

Plant gene products that have been described as `alcohol dehydrogenases' are surveyed and related to their CPGN nomenclature. Most are Zn-dependent medium chain dehydrogenases, including `classical' alcohol dehydrogenase (Adh1), glutathione-dependent formaldehyde dehydrogenase (Fdh1), cinnamyl alcohol dehydrogenase (Cad2), and benzyl alcohol dehydrogenase (Bad1). Plant gene products belonging to the short-chain dehydrogenase class should not be called alcohol dehydrogenases unless such activity is shown.     

Purification and properties of D-3-hydroxybutyrate dehydrogenase from Paracoccus denitrificans

D-3-Hydroxybutyrate dehydrogenase from Paracoccus denitrificans has been purified to near homogeneity. The enzyme was prepared using DEAE-cellulose chromatography, affinity chromatography on immobilized Cibacron blue (Matrex Gel Blue A) and gel permeation chromatography. The pure enzyme was obtained by chromatofocusing as the final isolation step. The purification procedure yielded the enzyme with a specific activity of about 100 units/mg protein. The enzyme is specific for D-3-hydroxybutyrate and NAD and it exhibits anomalous kinetics (hysteresis) at low enzyme and coenzyme concentrations. It is relatively stable in the presence of EDTA at pH 7–8 higer salt concentrations. D-3-Hydroxybutyrate dehydrogenase is a tetramer with a molecular weight of 130 000 ± 10 000, its isoelectric point equals 5.10 ± 0.05. The enzyme is applicable to the determination of acetoacetate and D-3-hydroxybutyrate concentrations.     

NAD- and co-substrate (GSH or factor)-dependent formaldehyde dehydrogenases from methylotrophic microorganisms act as a class III alcohol dehydrogenase

Abstract The methylotrophic yeasts, Hansenula polymorpha and Candida boidinii , and the methylotrophic Gram-negative bacteria, Paracoccus denitrificans and Thiobacillus versutus (but not Methylophaga marina ), contain NAD/GSH-dependent formaldehyde dehydrogenase when grown on C₁-compounds. The enzymes appeared to be similar to each other and to the mammalian counterparts with respect to substrate specificity, including the ability to act as an alcohol dehydrogenase class III. The Gram-positive bacteria, Amycolatopsis methanolica and Rhodococcus erythropolis , possess NAD/Factor-dependent formaldehyde dehydrogenase when grown on C₁-compounds or on C₁-unit-containing substrates, respectively. These enzymes also exhibit alcohol dehydrogenase class III activity. Thus, like the mammalian ones, methylotrophic formaldehyde dehydrogenases show dual substrate specificity, suggesting that this is an inherent property of the enzyme.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.     

UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases.

The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD(+)-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microhetergeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of approximately 26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Towards the molecular basis for the regulation of mitochondrial dehydrogenases by calcium ions

In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation. This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration. The other three enzymes are located within the mitochondrial matrix.While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not. This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium. FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme. In contrast, it is possible that the calcium sensitivity of the other dehydrogenases may involve separate calcium binding subunits.     

Alcohol dehydrogenase: multiplicity and relatedness in the solvent-producing clostridia

Abstract: Alcohol dehydrogenase (ADH) is a key enzyme for the production of butanol, ethanol, and isopropanol by the solvent-producing clostridia. Initial studies of ADH in extracts of several strains of Clostridium acetobutylicum and C. beijerinckii gave conflicting molecular properties. A more coherent picture has emerged because of the following results: (i) identification of ADHs with different coenzyme specificities in these species; (ii) discovery of structurally conserved ADHs (type 3) in three solvent-producing species; (iii) isolation of mutants with deficiencies in butanol production and restoration of butanol production with a cloned alcohol/aldehyde dehydrogenase gene; and (iv) resolution of various ' C. acetobutylicum ' cultures into four species. The three ADH isozymes of C. beijerinckii NRRL B592 have high sequence similarities to ADH-1 of Clostridium sp. NCP 262 (formerly C. acetobutylicum P262) and to the ADH domain of the alcohol/aldehyde dehydrogenase of C. acetobutylicum ATCC 824/DSM 792. The NADH-dependent activity of the ADHs from C. beijerinckii NRRL B592 and the BDHs from C. acetobutylicum ATCC 824 is profoundly affected by the pH of the assay, and the relative importance of NADH and NADPH to butanol production may be misappraised when NAD(P)H-dependent activities were measured at different pH values. The primary/secondary ADH of isopropanol-producing C. beijerinckii is a type-1 enzyme and is highly conserved in Thermoanaerobacter brockii (formerly Thermoanaerobium brockii ) and Entamoeba histolytica . Several solvent-forming enzymes (primary ADH, aldehyde dehydrogenase, and 3-hydroxybutyryl-CoA dehydrogenase) are very similar between C. beijerinckii and the species represented by Clostridium sp. NCP 262 and NRRL B643. The realization of such relationships will facilitate the elucidation of the roles of different ADHs because each type of ADH can now be studied in an organism most amenable to experimental manipulations.     

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.     

Regulation of methanol and methylamine dehydrogenases in Methylophilus methylotrophus

Abstract Methylophilus methylotrophus can use methylamine as sole source of carbon and nitrogen. Measurements of the specific activity of methylamine dehydrogenase (MNDH) in bacteria grown in batch or chemostat culture showed that MNDH was induced by methylamine and repressed when methanol or NH₄⁺ were provided as alternative carbon or nitrogen sources. The degree of repression varied with the growth conditions. Methanol dehydrogenase (MDH) was present in bacteria growtn on methylamine as sole carbon source, but the specific activity was low compared with that in bacteria grown on medium containing methanol, indicating that this enzyme is induced by methanol.