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1.
Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42 000 ± 1000 Da subunits,
with a native molecular weight of 174 000 ± 6000 Da. The optimal salt concentration, temperature, and pH for enzyme activity
were 2 M KCl or NaCl, 45°C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four
novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified.
Received: November 19, 1997 / Accepted: May 12, 1998 相似文献
2.
Ratnakar Vallabhaneni Louis M.T. Bradbury 《Archives of biochemistry and biophysics》2010,504(1):104-111
3.
该研究以‘铁观音’茶树为材料,同源克隆了茶树类胡萝卜素裂解双加氧酶基因CsCCD1和CsCCD4 (NCBI登录号分别为MH119136和MH119137)全长cDNA。序列分析结果显示,CsCCD1序列全长1 766 bp,包含1 641 bp开放阅读框(ORF),编码545个氨基酸,定位于细胞质中,不存在跨膜结构和信号肽;CsCCD4序列全长1 942 bp,包含1 842 bp ORF,编码612个氨基酸,不存在跨膜结构,但在N端具有叶绿体转运肽,定位于质体中。进化树分析显示,CsCCD1和CsCCD4与杜鹃聚为一类。荧光定量检测显示,CsCCD1和CsCCD4在叶、茎和花中表达量较高。在乌龙茶做青过程中,CsCCD1和CsCCD4基因都是从鲜叶到晒青表达量下调,而CsCCD1在一摇和二摇显著上调表达,在三摇后下降;CsCCD4只在一摇后上调表达,之后表达量迅速降低。推测CsCCD1和CsCCD4基因可能与乌龙茶做青香气品质形成关系密切。 相似文献
4.
Molecular characterisation of gibberellin 20-oxidases. Structure-function studies on recombinant enzymes and chimaeric proteins 总被引:1,自引:0,他引:1
Theo Lange Carsten Kegler Peter Hedden y L. Phillips Jan E. Graebe 《Physiologia plantarum》1997,100(3):543-549
Gibberellin (GA) 20-oxidases are multifunctional enzymes that catalyse reactions at an important branch point in the GA biosynthetic pathway. These enzymes oxidise the C-20 methyl group of a diterpene carboxylic acid precursor (e.g. GA12) to form an alcohol (in our case GA15-open lactone) and an aldehyde (GA24). The aldehyde is either oxidised to a tricarboxylic acid (GA25) or, with loss of carbon-20 and lactonisation, to a C19-GA (GA9). This branching is interesting to study, because C19-GA derivatives function as plant hormones in different tissues, whereas the C20-GA tricarboxylic acids have no known function. We have constructed chimaeric proteins by combining a GA 20-oxidase from immature seeds of Cucurbita maxima L., which produces mainly C-20 carboxylic acids, with a 20-oxidase from Marah macrocarpus immature seeds, which forms predominantly CC19-GAs. The cDNAs encoding these two very similar 20-oxidases were digested with restriction endonucleases Van 911. Bcl 1, and Bsa WI, and six chimaeric sequences were produced by recombination of the DNA fragments. The pCM1 -construct was obtained by exchanging nt 303–809 of the Cucurbita cDNA with the homologous DNA from the March 20-oxidase. In pCM2, pCM3, pCM4, pCM5 and pCM6, nt 810–992, nt 993–end, nt 303–992, nt 810–end, and nt 311–end were exchanged, respectively. All constructs were cloned in a pUC18 vector and functionally expressed in E. coli NM522 cells. GA 20-oxidase activity was detectable in cell-lysates from the transformed E. coli, but the extent and kind of conversion depended on the construct. Highest conversion of GA12was found with pCM1 and pCM3, one-tenth of this conversion was observed with pCM5 and pCM6, and one-hundredth was obtained with the hybrid proteins from pCM2 and pCM4. With pCM2 and pCM4, neither the C19-end product, GA9, nor the C20-end product, GA25-was formed. However, after transformation with constructs pCM1, pCM3, pCM5 or pCM6. GA9accounted for 30, 40, 60 and 90%, respectively, of the end products formed. Thus, the segments originating from M. macrocarpus conferred upon the chimaeric proteins an increasing ability to direct the biosynthetic flow into C19-GAs in this order. Although GA24is the immediate precursor, much less end products were formed by using this substrate. 相似文献
5.
Fluoranthene (Fla) is a high molecular weight polycyclic aromatic hydrocarbon that exerts hazardous effects on living organisms. An efficient Fla degrading bacterial consortium LP was enriched from an oil contaminated soil sample, with and without yeast extract as a supplement. Objective of the present study was to see if there was any differential effect of yeast extract addition on Fla degradation potential and aromatic ring dioxygenase expressing bacteria (ARDB) of the enrichments. Primary enrichment of the soil sample was carried out in minimal salt medium (MSM) added with 500 mg l−1 Fla and 0.05% yeast extract (YMSM). Secondary, tertiary and subsequent enrichments were prepared in YMSM and MSM after every sixteen days of incubation. Fla was efficiently degraded by YMSM enriched culture than MSM enriched culture. However, when MSM enrichment was incubated longer instead of further subculturings, it also degraded Fla efficiently. All three enrichments exhibited growth of bacterial colonies on Fla sprayed minimal agar plates however only YMSM enrichment showed clear zone forming bacterial colonies. A positive effect was observed of yeast extract on ARDB population of LP consortium. To our limited knowledge this is first time that effect of yeast extract on ARDB population was studied. 相似文献
6.
C. Hall D. McCallum A. Prescott R. Mithen 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,102(2-3):369-374
Fine mapping of the glucosinolate biosynthesis gene OHP, which regulates the conversion of 3-methylsulphinylpropyl to 3-hydroxypropyl glucosinolate, in an Arabidopsis thaliana Columbia × Landsberg erecta RI line population positioned the gene within 54 kb of DNA on chromosome IV. Sequence data identified a family of genes encoding
2-oxoglutarate-dependent dioxygenases in this region. A probe based on these genes co-segregated with ALK in Brassica oleracea,a gene regulating the synthesis of alkenyl glucosinolates. The reactions catalysed by the OHP and ALK enzymes utilise similar substrates and may have a common mechanism. Thus, these dioxygenases are prime candidates for controlling
the side chain modification of glucosinolates.
Received: 12 May 2000 / Accepted: 29 May 2000 相似文献
7.
The complexes [Mn(L1)(tcc)] (1), [Mn(L2)(tcc)] · H2O · 0.5CH3OH (2), [Mn(L3)(tcc)] · CH2Cl2 (3), [Mn(L4)(tcc)] · 1.5CH2Cl2 (4), [Mn(L5)(tcc)] (5), and (HN(C2H5)3)[Mn(L6)(tcc)] · CH2Cl2 (6) have been synthesized using the ligands HL1 (2-[(bis(pyridin-2-ylmethyl)amino)methyl]phenol), HL2 (2-[[((6-methylpyridin-2-yl)methyl)(pyridin-2-ylmethyl)amino]methyl] phenol), HL3 (2-[[((6-methylpyridin-2-yl)methyl)(pyridin-2-ylmethyl)amino]methyl]-4-nitrophenol), HL4 (2-[(bis(pyridin-2-ylmethyl)amino)methyl]-4-bromophenol), HL5 (2-[(bis(pyridin-2-ylmethyl)amino)methyl]-6-methoxyphenol) and H2L6 ([(bis(2-hydroxy-5-nitrobenzyl))(pyridin-2-ylmethyl)]amine) and characterized by X-ray crystallography, mass spectrometry, IR, UV-Vis spectroscopy, cyclic voltammetry, and elemental analysis. Compounds 1 and 6 crystallize in the monoclinic space groups P21/n and P21/c, respectively, whereas the crystal structures of complexes 2, 3, and 4 were solved in the triclinic space group . Complex 5 crystallizes in the orthorhombic space group P212121. Complexes 1-6 are structural related to the proposed active site of intradiol cleaving catechol dioxygenase exhibiting a distorted octahedral N3O3 (1-5) and N2O4 (6) donor set, respectively. Complexes 1-6 can be regarded as structural manganese analogous for substituted forms of iron-containing intradiol cleaving catechol dioxygenases, where the substrate tetrachlorocatechol (tcc) is asymmetrically bound to the metal center. 相似文献
8.
Gibberellins are ent-kaurene-derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent-kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent-copalyl diphosphate and ent-kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi. 相似文献
9.
Due to their selectivity biocatalytic hydroxylations catalysed by monooxygenases or dioxygenases arouse considerable interest. The most studied and distributed type of monooxygenases are cytochromes P-450, which are a supergene family of proteins that catalyse the oxidation of lipophilic compounds through the insertion of 1 oxygen atom of O2 into the substrate. For biocatalytic processes those cytochromes deserve attention, are involved in the catabolism of nutrients in microorganisms or in the metabolism of physiological substrates in higher eucaryotes. Mammalian cytochromes P-450, which catalyse the oxidation of xenobiotics (synthetic drugs, pesticides, hazardous waste compounds, are hardly suitable for this purpose. Processes with immobilized cytochromes P-450 can be excluded. The use of wild or genetically transformed strains of microorganisms is a suitable way. 相似文献
10.
Arabidopsis thaliana L. produces flavonoid pigments, i.e. flavonols, anthocyanidins and proanthocyanidins, from dihydroflavonol substrates. A small family of putative flavonol synthase (FLS) genes had been recognized in Arabidopsis, and functional activity was attributed only to FLS1. Nevertheless, other FLS activities must be present, because A. thalianafls1 mutants still accumulate significant amounts of flavonols. The recombinant FLSs and leucoanthocyanidin dioxygenase (LDOX) proteins were therefore examined for their enzyme activities, which led to the identification of FLS3 as a second active FLS. This enzyme is therefore likely responsible for the formation of flavonols in the ldox/fls1-2 double mutant. These double mutant and biochemical data demonstrate for the first time that LDOX is capable of catalyzing the in planta formation of flavonols. 相似文献