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1.
Masaru Wada Tomoko Matsumoto Shigeru Nakamori Mitsuru Sakamoto Michihiko Kataoka Ji-Quan Liu Nobuya Itoh Hideaki Yamada Sakayu Shimizu 《FEMS microbiology letters》1999,179(1):147-151
L-threo-3-Hydroxyaspartate dehydratase (L-threo-3-hydroxyaspartate hydro-lyase), which exhibited specificity for L-threo-3-hydroxyaspartate (K(m)=0.74 mM, V(max)=37.5 micromol min(-1) (mg protein)(-1)) but not for D-threo or D, L-erythro-3-hydroxyaspartate, was purified from a cell-free extract of Pseudomonas sp. T62. The activity of the enzyme was inhibited by hydroxylamine and EDTA, which suggests that pyridoxal 5'-phosphate and divalent cations participate in the enzyme reaction. The NH(2)-terminal amino acid sequence showed significant similarity to the Saccharomyces cerevisiae YKL218c gene product, a hypothetical threonine dehydratase. However, the purified enzyme showed no threonine dehydratase activity. 相似文献
2.
运用PCR技术从克雷伯氏菌的基因组中分别扩增得到了编码甘油脱水酶再激活酶α、β两个亚基的基因gdrA、gdrB。将gdrA、gdrB克隆至pMD-18T载体上,构建克隆载体pMD-gdrAB。经测序正确后,将gdrAB亚克隆至表达载体pET-28a( )上构建表达质粒pET-28gdrAB。利用双抗生素筛选法,将pET-28gdrAB与连有甘油脱水酶基因的表达载体pET-32gldABC在大肠杆菌菌株BL21(DE3)中共表达,鉴定了甘油脱水酶再激活酶的活性。 相似文献
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Wolfgang Buckel 《FEMS microbiology letters》1992,88(3-4):211-232
Abstract In amino acid fermenting anaerobic bacteria a set of unusual dehydratases is found which use 2-hydroxyacyl-CoA, 4-hydroxybutyryl-CoA or 5-hydroxyvaleryl-CoA as substrates. The extremely oxygen-sensitive 2-hydroxyacyl-CoA dehydratases catalysing the elimination of water from ( R )-lactyl-CoA to acryloyl-CoA or from ( R )-2-hydroxyglutaryl-CoA to glutaconyl-CoA contain iron-sulfur clusters as well as riboflavin and require additional activation by ATP. The dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA is catalysed by a moderately oxygen-sensitive enzyme also containing an iron-sulfur cluster and FAD. In all these reactions a non-activated C-H-bond at C3 has to be cleaved by mechanisms not yet elucidated. The dehydration of 5-hydroxyvaleryl-CoA to 4-pentenoyl-CoA, however, has been characterised as a redox process mediated by enzyme-bound FAD. Finally, an iron-sulfur cluster-containing but pyridoxal-phosphate-independent l -serine dehydratase is described. 相似文献
5.
Sakayu Shimizu Keijiro Miyata Yoshiki Tani Koichi Ogata 《Bioscience, biotechnology, and biochemistry》2013,77(3):615-619
A new process has been described for the preparation of coenzyme A of high purity from the cultured broth of Brevibacterium ammoniagenes IFO 12071. The product was obtained in a high yield by the use of Duolite S–30, charcoal, and Dowex 1×2, and identified chemically and enzymatically. This method is simple, rapid, and compact, requires no special equipment, and has been shown to be adaptable for preparing large amounts of highly pure coenzyme A. 相似文献
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The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Porphobilinogen synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. In the present study, a variety of new substrate analogs and reaction intermediate analogs were synthesized, which were used as probes for studying the active site of rat porphobilinogen synthase. The compounds 1, 3, 6, 9, 14, 16, and 28 were found to be competitive inhibitors of rat porphobilinogen synthase with inhibition constants ranging from 0.96 to 73.04 mM. Compounds 7, 10, 12, 13, 15, 17, 18, and 26 were found to be irreversible enzyme inhibitors. For irreversible inhibitors, loose-binding inhibitors were found to give stronger inactivation. The amino group and carboxyl group of the analogs were found to be important for their binding to the enzyme. This study increased our understanding of the active site of porphobilinogen synthase. 相似文献
8.
1,3-丙二醇是一种重要的化工原料,其生物法生产的研究逐渐受到的关注。研究以弗氏柠檬酸菌的总DNA为模板,通过PCR分别扩增出约1.8kb(dhaF)和0.4kb(dhaG)的两个基因片段分别编码甘油脱水酶激活因子大、小亚基, 连接于pMD-18T载体,测序分析显示与GenBank中相关基因的相似性最高为86%。将两基因以多顺反子的方式与pSE380连接构建表达载体,并在大肠杆菌中进行高效表达,表达量占总蛋白的30%。将高效表达的激活因子用金属亲合层析和分子筛进行了纯化,得到电泳纯级的甘油脱水酶激活因子,SDS-PAGE分析显示:大、小亚基分子量约为63kDa和12kDa;非变性胶分析显示:全酶的分子量约为150kDa,经扫描分析推测甘油脱水酶激活因子很有可能是以α2β2方式结合的。以弗氏柠檬酸菌甘油脱水酶为研究对象,进行激活实验,结果证实该激活因子具备甘油脱水酶激活因子的功能,为进一步阐明甘油脱水酶的激活机制及1,3-丙二醇的高效生产奠定了基础。 相似文献
9.
Christian S. Lentz Dagmar Stumpfe Juergen Bajorath Michael Famulok Achim Hoerauf Kenneth M. Pfarr 《Bioorganic & medicinal chemistry letters》2013,23(20):5558-5562
Substituted benzimidazoles of the wALADin1-family have recently been identified as a new class of species-selective inhibitors of delta-aminolevulinic acid dehydratase (ALAD) from Wolbachia endobacteria of parasitic filarial worms. Due to its Wolbachia-dependent antifilarial activity, wALADin1 is a starting point for the development of new drugs against filarial nematodes. We now present several other chemotypes of ALAD inhibitors that have been identified based upon their molecular similarity to wALADin1. A tricyclic quinoline derivative (wALADin2) with a different inhibitory mechanism and improved inhibitory potency and selectivity may represent an improved drug lead candidate. 相似文献
10.
Xu-De Wang Xian-En Zhang Yong-Chao Guo Zhi-Ping Zhang Zhu-An Cao Ya-Feng Zhou 《Biotechnology letters》2009,31(5):711-717
The gdh and gdhr genes, encoding B12-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the β-subunit was lost during GDH purification when co-expressing α, β and γ subunit. This was overcome by fusing
the β-subunit to α- or γ-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties
of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein
GDHALB/C, constructed by linking the N-terminal of β-subunit to the C-terminal of α subunit through a (Gly4Ser)4 linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based
inactivation by glycerol during catalysis and could be reactivated by GDHR.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
弗氏柠檬酸菌甘油脱水酶基因在大肠杆菌中的克隆和表达 总被引:4,自引:0,他引:4
以弗氏柠檬酸菌(Citrobacter freundii)基因组DNA为模板,通过PCR得到甘油脱水酶(glycerol dehydratase)基因dhaB、dhaC、dhaE,克隆到表达载体pSE380上,得到重组质粒pSn-dhaBCE。将此重组质粒转化到E.coli JM109中,重组菌株SDS-PAGE结果显示有明显的61kD、22kD、16kD三条特异性蛋白条带出现。重组菌株经诱导表达,酶活力为11.59U/mL。 相似文献
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重组大肠杆菌生物转化甘油生产3-羟基丙酸 总被引:1,自引:0,他引:1
目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。 相似文献
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Abstract The threonine dehydratase activity of several yeast species was examined. Of 14 species tested, Candida maltosa, Pichia guilliermondii, Pichia pinus and Yarrowia lipolytica were found to produce an enzyme whose activity was increased about 20-fold in the presence of phosphate ions. It is suggested that phosphate affects the allosteric properties of the enzyme and leads, thereby, to an alteration of the binding of threonine (substrate), isoleucine (negative effector), and valine (positive effector) as well as to an alteration of the effects of temperature on the enzyme including changes in temperature optimum, energy of activation, and heat inactivation. 相似文献
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Suresh Bhosale Deepa Kshirsagar Prashant Pawar Tulsiram Yeole Dilip Ranade 《FEMS microbiology letters》1995,127(1-2):151-155
Abstract 5-Aminolevulinic acid dehydratase from the archaebacterium Methanosarcina barken resembles the mammalian and yeast enzymes in its activation by Zn2+ , whereas its activation by K+ resembles the characteristic of bacterial enzymes. This enzyme is activated with Ni2+ which is a component of F430 , a cofactor present mainly in methanogens. The M r of 280000 for the native enzyme and 30 000 ± 2000 for the individual subunit suggest that the enzyme is composed of eight apparently indentical subunits similar to mammalian and yeast enzymes. The enzyme has two pH optima, at 8.5 and 9.4. Higher levels of 5-aminolevulinic acid dehydratase in acetate-grown cells suggest the possibility that regulation and control of this enzyme could be different on various growth substrates. 相似文献
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克雷伯氏菌甘油脱水酶基因在大肠杆菌中的克隆与表达 总被引:5,自引:2,他引:5
利用PCR技术从克雷伯氏菌(Klebsiella pneumoniae ATCC49790)总DNA中扩增得到甘油脱水酶(glycerol dehydratase,DHAB)基因的DNA片段,并将其连接到表达质粒pSE380,携带有重组质粒pSE-dhaB的大肠杆菌JM109实现了dhaB基因的表达;对含有dhaB工程菌进行表达研究,表明工程菌在37℃,以1.0mmol/L IPTG诱导5h酶活力即达到1164.14u/L,比野生菌酶活力(168.69U/L)提高了6.9倍。 相似文献
16.
Takamasa Tobimatsu Tsuneo Nishiki Masaya Morimoto Ryou Miyata Tetsuo Toraya 《Archives of microbiology》2009,191(3):199-206
Coenzyme B12-dependent diol and glycerol dehydratases are isofunctional enzymes, which catalyze dehydration of 1, 2-diols to produce corresponding
aldehydes. Although the two types of dehydratases have high sequence homology, glycerol dehydratase is a soluble cytosolic
enzyme, whereas diol dehydratase is a low-solubility enzyme associated with carboxysome-like polyhedral organelles. Since
both the N-terminal 20 and 16 amino acid residues of the β and γ subunits, respectively, are indispensable for the low solubility
of diol dehydratase, we constructed glycerol dehydratase-based chimeric enzymes which carried N-terminal portions of the β
and γ subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-specific
N-terminal 34 and 33 amino acid residues of the β and γ subunits, respectively, was not enough to lower the solubility of
glycerol dehydratase. A chimeric enzyme which carries the low homology region (residues 35–60) of the diol dehydratase β subunit
in addition to the diol dehydratase-specific extra-regions of β and γ subunits showed low solubility comparable to diol dehydratase,
although its hydropathy plot does not show any prominent hydrophobic peaks in these regions. It was thus concluded that short
N-terminal sequences are sufficient to change the solubility of the enzyme. 相似文献
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1,3-丙二醇是一种重要的化工原料,其生物法生产的研究越来越受到广泛的关注。以克雷伯氏菌的总DNA为模板,通过PCR分别扩增出约1.8kb的gdrA和0.4kb的gdrB的两个基因片段,随后,将此两基因以多顺反子的方式与pSE380相连构建表达载体,并在大肠杆菌中进行了高效表达,表达量约占总蛋白的30%。将高效表达的激活因子用金属亲合层析和分子筛进行了纯化,得到电泳纯级的激活因子,SDS-PAGE分析显示:大、小亚基分子量约为64kDa和12kDa;非变性胶分析显示:全酶的分子量约为150kDa,扫描分析激活因子是以勉&方式结合的。以克雷伯氏菌甘油脱水酶为研究对象,进行激活实验,结果证实该激活因子具备甘油脱水酶激活因子的功能。该研究为进一步阐明甘油脱水酶的激活机制及1,3一丙二醇的高效生产奠定了基础。 相似文献
19.
Chin S. Chang Shigeru Sassa Darell Doyle 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,797(3):297-301
Rabbit antibody directed to homogeneously purified mouse liver δ-aminolevulinic acid dehydratase cross-reacted with the enzyme in erythrocytes, spleen, kidney and brain in the mouse. The antibody also cross-reacted with the enzyme in the rat, hamster and gerbil, but not in the rabbit, guinea pig, cattle, chick embryo, and human. In contrast, rabbit antibody against the human enzyme partially recognized the monkey enzyme, but not the enzyme in the other species. The species specificity of δ-aminolevulinic acid dehydratase in this study was consistent with the phylogenetic evolution of the species examined. 相似文献
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Pakhomova S Buck J Newcomer ME 《Protein science : a publication of the Protein Society》2005,14(1):176-182
The structure of retinol dehydratase (DHR) from Spodoptera frugiperda, a member of the sulfotransferase superfamily, in complexes with the inactive form of the cofactor PAP 3'-phosphoadenosine 5'-phosphate (PAP) and (1) the product of the reaction with retinol anhydroretinol (AR), (2) the retinoid inhibitor all-trans-4-oxoretinol (OR), and (3) the potent steroid inhibitor androsterone (AND) have been determined and compared to the enzyme complex with PAP and retinol. The structures show that the geometry of the active-site amino acids is largely preserved in the various complexes. However, the beta-ionone rings of the retinoids are oriented differently with respect to side chains that have been shown to be important for the enzymatic reaction. In addition, the DHR:PAP:AND complex reveals a novel mode for steroid binding that contrasts significantly with that for steroid binding in other sulfotransferases. The molecule is displaced and rotated approximately 180 degrees along its length so that there is no acceptor hydroxyl in close proximity to the site of sulfate transfer. This observation explains why steroids are potent inhibitors of retinol dehydratase activity, rather than substrates for sulfonation. Most of the steroid-protein contacts are provided by the alpha-helical cap that distinguishes this member of the superfamily. This observation suggests that in addition to providing a chemical environment that promotes the dehydration of a sulfonated intermediate, the cap may also serve to minimize a promiscuous sulfotransferases activity. 相似文献