植物3-磷酸甘油醛脱氢酶的多维本质

3-磷酸甘油醛脱氢酶(GAPDH)作为一种糖酵解蛋白在糖酵解的能量产生中发挥着重要作用。它通常作为一种模式蛋白用于蛋白和酶的分析,也可以用作研究基因表达量的内在对照。然而,最近的相关研究表明,真核及原核生物的3-磷酸甘油醛脱氢酶实际上存在着一种多维本质,研究证明它在DNA修复、细胞凋亡、核RNA输出、及其在细胞周期中都发挥着重要的作用。尽管该酶在植物中的研究不如在哺乳动物中的深入,但研究已经陆续证明,3-磷酸甘油醛脱氢酶在植物中同样具有许多未被发现的功能,目前已经报道该酶在厌氧、热激、伤害以及能量供应中可能发挥着重要作用。本文旨在就国内外对于该酶在植物中的研究作一总结论述,以期推进科学界对它的更深入认识和研究。     

甲氰味胍能刺激降钙素释放

甲氰咪胍(cimetidine)是一种组织胺H_2受体拮抗剂,用于治疗胃液分泌过多。最近,一些实验提示,甲氰咪胍能抑制甲状旁腺激素(PTH)的分泌以及甲状旁腺组织中cAMP的产生。给正常人或病人用甲氰味胍,能使血清中PTH的免疫反应降低。作者实验室进一步观察了甲氰咪胍是否也影响降钙素(CT)的释放。从幼年大鼠制备甲状腺-甲状旁     

利用酶清除室内甲醛气体的新方法

甲醛脱氢酶是一种可以将甲醛进行转换的氧化还原酶,利用改质甲醛脱氢酶对于甲醛的专一反应性可达到去除甲醛气体的效果.根据密闭空间测试甲醛去除效率,在10min 内可去除空间中85% 的甲醛气体.经过现场测试后发现,将甲醛去除器置放在含有福尔马林的储藏室中可在一周内降低70% 的甲醛浓度,两周后可将甲醛浓度降至安全标准以下.结果显示甲醛去除器不仅能够去除实验室中密闭空间内的甲醛气体,在一般布满甲醛气体的空间中依然可以在短时间内去除空间中的甲醛气体,避免人体受到甲醛的危害.     

痢疾杆菌体内诱导基因筛选方法的建立

分别用氯霉素乙酰转移酶基因(cat)和天冬氨酸半醛脱氢酶基因(asd)作为报告基因,用小鼠肺感染模型和HeLa细胞侵袭模型来试验筛选痢疾杆菌体内诱导基因的可行性。结果表明,以asd为报告基因,用HeLa细胞侵袭试验作为筛选模型,可成功地用于筛选痢疾杆菌的体内诱导基因。     

鱼腥藻PCC7120中琥珀酸半醛脱氢酶的初步表征及结构分析

蓝藻琥珀酸半醛脱氢酶催化琥珀酸半醛到琥珀酸的转化,使得蓝藻的三羧酸循环变得完整。BLAST序列分析预测鱼腥藻PCC7120中all3556基因编码琥珀酸半醛脱氢酶。为了证实all3556基因编码蛋白的催化功能,构建了p ET28a-all3556表达质粒,并在大肠杆菌BL21(DE3)菌体中进行重组蛋白诱导表达;利用镍亲和层析方法对all3556蛋白进行了分离纯化。酶动力学测试表明all3556蛋白是一个NADP+-依赖型的琥珀酸半醛脱氢酶。生物信息学分析发现all3556蛋白和其他来源的琥珀酸半醛脱氢酶的氨基酸序列具有一定的同源性,在催化中心的氨基酸残基高度保守。     

微生物组大数据管理与分析

高通量技术的迅猛发展促使微生物生态学研究获得了重大突破,掀起了元基因组学(Metagenomics)研究的热潮。元基因组学通常被定义为对未培养的环境样本中微生物群体的DNA序列分析。随着微生物组学数据的日益剧增,微生物大数据的高效管理与分析越来越受到研究者的关注。如何从海量的微生物组数据中挖掘出具有科研价值的数据信息并应用于实际问题成为当前的研究热点。目前已有很多计算生物学程序工具及数据库用于元基因组数据的分析与管理。本文主要综述了随着高通量测序技术的进步,国际上主要的微生物组计划及微生物组数据平台,如人类微生物组项目(human microbiome project,HMP)、地球微生物组项目(earth microbiome project,EMP)、欧盟的肠道微生物组计划(metagenomics of human intestinal tract,MetaHIT)、MG-RAST、i Microbe、整合微生物组(integration microbial genomes,IMG)以及EBI Metagenomics等;介绍了微生物数据分析的主要流程与工具;提出了建设多源异构的微生物生态数据管理与分析系统的必要性。     

甜高粱琥珀酸半醛脱氢酶SbSSADH基因克隆及原核表达

γ-氨基丁酸(γ-aminobutyric acid,GABA)代谢旁路是TCA循环的一个代谢支路,广泛存在于动植物和微生物中,植物GABA代谢旁路与植物生物和非生物胁迫响应相关。琥珀酸半醛脱氢酶(SSADH)是GABA旁路的一个关键酶,对GABA途径在生物体内发挥功能起着至关重要的作用。通过检索Maize GDB和Gramene数据库获得甜高粱Sb SSADH基因信息,利用同源克隆的方法获得了甜高粱Sb SSADH基因。序列分析结果表明,Sb SSADH基因与玉米、甘蔗在核酸序列的相似度为94.77%,但前端约200 bp的信号肽部分差异较大。通过以p ET-28a为表达载体构建Sb SSADH原核表达系统,Rosetta菌株为表达菌,表达出约50 k D的SSADH蛋白,将纯化的蛋白进行酶活分析,纯化蛋白具有琥珀酸半醛脱氢酶活性,并且受酶抑制剂ATP和AMP的抑制。     

海因酶法制备D-对羟基苯甘氨酸的研究进展

D-对羟基苯甘氨酸(D-HPG)主要用于合成β-内酰胺类半合成抗生素,是国内最紧缺的医药中间体之一。微生物酶法是目前获得光学纯D-HPG的重要途径,微生物中起催化作用的主要是D-海因酶和N-氨甲酰水解酶。文章综述了产酶微生物的来源,酶的理化性质,以及培养条件的优化、基因工程、酶的固定化技术生产D-HPG的研究进展。     

两种滨藜甜菜碱醛脱氢酶基因的克隆及序列分析

甜菜碱醛脱氢酶(Betaine aldehyde dehydrogenase,BADH)对非生物胁迫下植物渗透调节物质的合成和积累具有重要作用。分别从异苞滨藜和鞑靼滨藜两种盐生植物中分离到了BADH基因。序列分析表明,BADH全长均为1 507bp,编码501个氨基酸,两种BADH序列具有较高的相似性。甜菜碱醛脱氢酶的克隆为植物的基因转化及其功能分析奠定了基础。     

丙烯酰胺的微生物生产

丙烯酰胺的微生物生产 丙烯酰胺(Acrylamide,CH=CHCONH)合成水溶性聚合物如聚丙烯酰胺,或其共聚物,很有实用价值,可用作土壤改良剂、絮凝剂、黏合剂、造纸工业的纸张增强剂以及用于涂料等等,作为一种吸附剂可用于核燃料生产,如吸附海水中所含的铀(UO).     

Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases.

Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.     

Activities of the enzymes of formaldehyde catabolism in recombinant strains of <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Activities of the enzymes of formaldehyde (FA) catabolism in recombinant strains of the methylotrophic yeast Hansenula polymorpha overproducing NAD⁺- and glutathione-dependent formaldehyde dehydrogenase (FADH) were studied under different cultivation conditions and at elevated FA content. Southern dot-blot analysis confirmed the presence of six to eight copies of the target FLD1 gene in stable recombinant clones of H. polymorpha. Under certain cultivation conditions, the transformants resistant to elevated FA concentrations were shown to produce FADH and other bioanalytically important enzymes: formate dehydrogenase, alcohol dehydrogenase, alcohol oxidase, and formaldehyde reductase. The optimal cultivation conditions for recombinants were determined, resulting in maximum synthesis of FADH: methanol as a carbon source, methylamine as a nitrogen source, FA as an inducer, temperature of 37°C, and cells in the early exponential phase of growth.     

The nicotinamide adenine dinucleotide-binding site of chicken liver xanthine dehydrogenase. Evidence for alteration of the redox potential of the flavin by NAD binding or modification of the NAD-binding site and isolation of a modified peptide

Affinity labeling of the NAD-binding site of chicken liver xanthine dehydrogenase by 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA) caused spectral perturbation around 450 nm in the same way as NAD. Reductive titration with xanthine of native xanthine dehydrogenase in the presence of NAD showed that redox potentials of the FAD/FADH. and FADH./FADH2 couples were shifted positive by NAD binding to the enzyme. The redox potentials of these couples were also shifted to some extent by modification of the NAD-binding site with 5'-FSBA. These results provide further evidence that binding of NAD to chicken liver xanthine dehydrogenase modulates the reactivity of the enzyme by shifting the redox potential of FAD. Proteolytic cleavage of the [14C]-5'-FSBA-modified enzyme yielded several domain peptides, only one of which contained radioactivity. The isolated radioactive peptide was further digested with Staphylococcus aureus protease and the 14C-labeled peptide was purified by two steps of high performance liquid chromatography. The amino acid sequence of the peptide was determined, and a reactive tyrosine residue was identified.     

Differences in redox and kinetic properties between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase

Reductive titrations of a NAD-dependent type (type-D) and an O2-dependent type (type-O) of rat liver xanthine dehydrogenase showed that only the type-D enzyme formed a pronounced stable FAD semiquinone (FADH*). The FAD semiquinone was less stabilized in the presence of NAD. The Vmax value for xanthine-NAD activity of type-D enzyme was close to that for xanthine-O2 activity of type-O enzyme, while the Vmax value for xanthine-O2 activity of type-D enzyme was about one-fourth of that of type-O enzyme. The Km value for O2 of type-D enzyme was about five times as large as that of type-O enzyme. The absorbance spectrum of type-D enzyme during turnover with xanthine and O2 as substrates showed a considerable amount of FADH* formation, but that with xanthine and NAD as substrates showed only a negligible one. Low xanthine-O2 activity of type-D enzyme, as compared with that of type-O enzyme, seems to be explained by the conformational change occurring in conversion from type-O to type-D enzyme, which results in different reactivity of FAD to molecular oxygen and a higher fraction of FADH* during turnover. The binding of NAD may possibly increase the fraction of FADH2, resulting in a Vmax value of xanthine-NAD activity almost as high as that of xanthine-O2 activity of type-O enzyme.     

FAD requirement for the reduction of coenzyme F420 by formate dehydrogenase from Methanobacterium formicicum.

The partial purification of the formate dehydrogenase from cell-free extracts of Methanobacterium formicicum decreased the rate of coenzyme F420 reduction 175-fold relative to the rate of methyl viologen reduction. FAD, isolated from this organism, reactivated the coenzyme F420-dependent activity of purified formate dehydrogenase and restored the activity ratio (coenzyme F420/methyl viologen) to near that in cell-free extracts. Neither flavin mononucleotide nor FADH2 replaced FAD. The reduced form of FAD inhibited the reactivation of coenzyme F420-dependent formate dehydrogenase activity by the oxidized form. The results suggest that native formate dehydrogenase from Methanobacterium formicicum contains noncovalently bound FAD that is required for coenzyme F420-dependent activity.     

Coordinated production and utilization of FADH2 by NAD(P)H-flavin oxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase

4-Hydroxyphenylacetate (4HPA) 3-monooxygenase (HpaB) is a reduced flavin adenine dinucleotide (FADH(2)) utilizing monooxygenase. Its cosubstrate, FADH(2), is supplied by HpaC, an NAD(P)H-flavin oxidoreductase. Because HpaB is the first enzyme for 4HPA metabolism, FADH(2) production and utilization become a major metabolic event when Escherichia coli W grows on 4HPA. An important question is how FADH(2) is produced and used, as FADH(2) is unstable in the presence of free O(2). One solution is metabolic channeling by forming a transitory HpaB-HpaC complex. However, our in vivo and in vitro data failed to support the interaction. Further investigation pointed to an alternative scheme for HpaB to sequester FADH(2). The intracellular HpaB concentration was about 122 microM in 4HPA-growing cells, much higher than the total intracellular FAD concentration, and HpaB had a high affinity for FADH(2) (K(d) of 70 nM), suggesting that most FADH(2) is bound to HpaB in vivo. The HpaB-bound FADH(2) was either used to rapidly oxidize 4HPA or slowly oxidized by O(2) to FAD and H(2)O(2) in the absence of 4HPA. Thus, HpaB's high intracellular concentration, its high affinity for FADH(2), its property of protecting bound FADH(2) in the absence of 4HPA, and its ability to rapidly use FADH(2) to oxidize 4HPA when 4HPA is available can coordinate FADH(2) production and utilization by HpaB and HpaC in vivo. This type of coordination, in responding to demand, for production and utilization of labile metabolites has not been reported to date.     

Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH(2))-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH(2) in vitro, HpaB was able to use FADH(2) and O(2) for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH(2) for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH(2)-utilizing monooxygenase. FADH(2) generated by Fre was rapidly oxidized by O(2) to form H(2)O(2) in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH(2) and transitorily protected it from rapid autoxidation by O(2). When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O(2) for FADH(2) utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH(2) produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH(2) was autoxidized by O(2), causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH(2)-utilizing monooxygenase that uses FADH(2) as a substrate rather than as a cofactor.     

Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives

Native DNA photolyase, as isolated from Escherichia coli, contains a neutral flavin radical (FADH.) plus a pterin chromophore (5,10-methenyltetrahydropteroylpolyglutamate) and can be converted to its physiologically significant form by reduction of FADH. to fully reduced flavin (FADH2) with dithionite or by photoreduction. Either FADH2 or the pterin chromophore in dithionite-reduced native enzyme can function as a sensitizer in catalysis. Various enzyme forms (EFADox, EFADH., EFADH2, EPteFADox, EPteFADH., EPteFADH2, EPte) containing stoichiometric amounts of FAD in either of its three oxidation states and/or 5,10-methenyltetrahydrofolate (Pte) have been prepared in reconstitution experiments. Studies with EFADox and EPte showed that these preparations retained the ability to bind the missing chromophore. The results suggest that there could be considerable flexibility in the biological assembly of holoenzyme since the order of binding of the enzyme's chromophores is apparently unimportant, the binding of FAD is unaffected by its redox state, and enzyme preparations containing only one chromophore are reasonably stable. The same catalytic properties are observed with dithionite-reduced native enzyme or EFADH2. These preparations do not exhibit a lag in catalytic assays whereas lags are observed with preparations containing FADox or FADH. in the presence or absence of pterin. Photochemical studies show that these lags can be attributed to enzyme activation under assay conditions in a reaction involving photoreduction of enzyme-bound FADox or FADH. to FADH2. EPte is catalytically inactive, but catalytic activity is restored upon reconstitution of EPte with FADox. The results show that pterin is not required for dimer repair when FADH2 acts as the sensitizer but that FADH2 is required when dimer repair is initiated by excitation of the pterin chromophore. The relative intensity of pterin fluorescence in EPte, EPteFADH., EPteFADox, or EPteFADH2 has been used to estimate the efficiency of pterin singlet quenching by FADH. (93%), FADox (90%), or FADH2 (58%). Energy transfer from the excited pterin to flavin is energetically feasible and may account for the observed quenching of pterin fluorescence and also explain why photoreduction of FADox or FADH. is accelerated by the pterin chromophore. An irreversible photobleaching of the pterin chromophore is accelerated by FADH2 in a reaction that is accompanied by a transient oxidation of FADH2 to FADH.. Both pterin bleaching and FADH2 oxidation are inhibited by substrate.     

Electron paramagnetic resonance properties and oxidation-reduction potentials of the molybdenum,flavin, and iron-sulfur centers of chicken liver xanthine dehydrogenase

The oxidation-reduction potentials of the various prosthetic groups in the native and desulfo forms of chicken liver xanthine dehydrogenase, determined by potentiometric titration in 0.05 m potassium phosphate buffer, pH 7.8, are: Mo(VI)/Mo(V) (native), ?357 mV; Mo(VI)/Mo(V) (desulfo), ?397 mV; Mo(V)/Mo(IV) (native), ?337 mV; Mo(V)/Mo(IV) (desulfo), ?433 mV; FAD/FADH · ?345 mV; FADH · FADH₂, ? 377 mV; (Fe/S)I_ox/(Fe/S)I_red, ?280 mV; (Fe/S)II_ox/(Fe/S)II_red, ? 275 mV. Titration at pH 6.8 revealed that the Mo and FAD centers but not the Fe/S centers are in prototropic equilibrium. Spectroscopic studies on the native and deflavinated enzymes show that environment of the flavin in xanthine dehydrogenase differs from that in bovine milk xanthine oxidase.     

Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro

DNA photolyases repair cyclobutadipyrimidines (Pyr()Pyr) in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor. During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity. Previous nanosecond flash photolysis studies [Heelis, P.F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] indicated that excitation of FADH. either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH. quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme. Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction. We found that W306F mutation abolished photoreduction of FADH. without affecting the excited-state properties of FADH. or the substrate binding (KA approximately 10(9) M-1) of the enzyme. The specificity constant (kcat/km) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH. is an essential step for photorepair by photolyase in vitro. Chemical reduction of FADH. of the mutant enzyme restored the specificity constant to the wild-type level.