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1.
Valencia E Larroy C Ochoa WF Parés X Fita I Biosca JA 《Journal of molecular biology》2004,341(4):1049-1062
The crystal structure of Saccharomyces cerevisiae ScAdh6p has been solved using the anomalous signal from the two zinc atoms found per subunit, and it constitutes the first structure determined from a member of the cinnamyl alcohol dehydrogenase family. ScAdh6p subunits exhibit the general fold of the medium-chain dehydrogenases/reductases (MDR) but with distinct specific characteristics. In the three crystal structures solved (two trigonal and one monoclinic), ScAdh6p molecules appear to be structural heterodimers composed of one subunit in the apo and the second subunit in the holo conformation. Between the two conformations, the relative disposition of domains remains unchanged, while two loops, Cys250-Asn260 and Ile277-Lys292, experience large movements. The apo-apo structure is disfavoured because of steric impairment involving the loop Ile277-Lys292, while in the holo-holo conformation some of the hydrogen bonds between subunits would break apart. These suggest that the first NADPH molecule would bind to the enzyme much more tightly than the second. In addition, fluorimetric analysis of NADPH binding demonstrates that only one cofactor molecule binds per dimer. Therefore, ScAdh6p appears to function according to a half-of-the-sites reactivity mechanism, resulting from a pre-existing (prior to cofactor binding) tendency for the structural asymmetry in the dimer. The specificity of ScAdh6p towards NADPH is mainly due to the tripod-like interactions of the terminal phosphate group with Ser210, Arg211 and Lys215. The size and the shape of the substrate-binding pocket correlate well with the substrate specificity of ScAdh6p towards cinnamaldehyde and other aromatic compounds. The structural relationships of ScAdh6p with other MDR structures are analysed. 相似文献
2.
Sulzenbacher G Alvarez K Van Den Heuvel RH Versluis C Spinelli S Campanacci V Valencia C Cambillau C Eklund H Tegoni M 《Journal of molecular biology》2004,342(2):489-502
In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols. The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C(3). YqhD is a dimer of 2x387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain. The crystals contain two dimers in the asymmetric unit. While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites. The active site contains a Zn atom, as verified by EXAFS on the crystals. The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6. Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2). These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions. 相似文献
3.
牛肝辅酶Ⅱ依赖性视黄醇脱氢酶cDNA的克隆及组织表达 总被引:3,自引:0,他引:3
迄今为止的研究证明 ,维生素A亦称视黄醇(retinol)的生理功能是通过其两步氧化代谢产物视黄醛与视黄酸 (亦称维甲酸 )来完成的 .视黄醛通过其光学异构体 1 1 顺式视黄醛与视觉细胞内的视蛋白 (opsin)结合组成视色素 .感光时 ,1 1 顺式视黄醛转变成全反式视黄醛从视蛋白脱落 ,这一过程同时传导到大脑产生视觉[1 ] .全反式维甲酸 (all transretinoicacid)则通过与其在核内受体 (RARα ,β ,γ)结合调节基因的转录来发挥其许多重要的生理功能 ,包括正常胚胎的发育 ,形态、神经系统的形成 ,成体动物的生长、发育、繁殖等 ,并通过调解组织及… 相似文献
4.
即时浸酸显著提高滞育性家蚕卵辅酶Ⅰ和Ⅱ含量 总被引:1,自引:0,他引:1
即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 提高了其呼吸耗氧量, 抑制了山梨醇积累。本研究利用HPLC法测定了家蚕滞育卵和5 min即时浸酸滞育性卵中辅酶Ⅰ和Ⅱ含量。结果表明: 产下后24-72 h, 家蚕滞育卵中NAD, NADH, NADP和NADPH含量分别下降了30%, 37%, 50%和4%; 而即时浸酸滞育性卵中分别增加了77%, 46%, 142%和241%。不过, 即时浸酸并未显著改变滞育性家蚕卵中NADH/NAD和NADPH/NADP比值。据此推测, 即时浸酸提高滞育性家蚕卵辅酶Ⅰ含量与其呼吸耗氧量增加有关; 即时浸酸显著提高辅酶Ⅱ含量与山梨醇积累抑制无关, 而主要与生物合成加强有关。 相似文献
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从牛的肝脏中快速抽提总RNA,根据GenBank已发表NADP(H)-依赖的视黄醇脱氢酶基因(NRDR)的cDNA序列,设计并合成特异引物,利用cDNA末端快速扩增(RACE)方法和反转录-聚合酶链式反应(RT-PCR),得到牛肝内的NRDR cDNA的全长序列。经测序证实,牛肝NRDR的全长cDNA序列为1266bp,其开放读码框架在24~806bp,编码260个氨基酸(GenBank登录号:AF487454)。根据NRDR基因推导出的氨基酸序列与人、鼠、兔有高度同源性,并含有SDR超家族成员的两个高度保守的模序,在其C-端含有过氧化物酶体的靶向序列为SHL。结果表明,牛的NRDR应属于过氧化物酶体内SDR超家族成员并在维甲酸合成的限速步骤起作用的酶,也为维甲酸合成的传统通路提供一个补充。 相似文献
7.
Tomita T Fushinobu S Kuzuyama T Nishiyama M 《Biochemical and biophysical research communications》2005,334(2):613-618
For better understanding of the coenzyme specificity in NAD-dependent MDH (tMDH) from Thermus flavus AT-62, we determined the crystal structures of tMDH-NADP(H) complex at maximally 1.65 A resolution. The overall structure is almost the same as that of the tMDH-NADH complex. However, NADP(H) binds to tMDH in the reverse orientation, where adenine occupies the position near the catalytic center and nicotinamide is positioned at the adenine binding site of the tMDH-NADH complex. Consistent with this, kinetic analysis of the malate-oxidizing reaction revealed that NADP(+) inhibited tMDH at high concentrations. This has provided the first evidence for the alternative binding mode of the nicotinamide coenzyme, that has pseudo-symmetry in its structure, in a single enzyme. 相似文献
8.
This study describes the development and application of a bioaffinity chromatographic system for the one-step purification of an NADP(+)-dependent secondary alcohol dehydrogenase from the obligate anaerobe, Thermoanaerobacter brockii (TBADH, EC 1.1.1.2). The general approach is based upon improving the selectivity of immobilized cofactor derivatives (general ligand approach to bioaffinity chromatography) through using soluble enzyme-specific substrate analogues in irrigants to promote biospecific adsorption (the kinetic locking-on tactic). Specifically, the following is described: Evaluation of 8'-azo-linked, C(8)-linked, N(1)-linked, and N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of TBADH; evaluation of 2', 5'-ADP as a stripping ligand for TBADH bioaffinity purifications using an 8'-azo-linked immobilized NADP(+) derivative in the locking-on mode; and application of the developed bioaffinity chromatographic system to the purification of TBADH from a crude cellular extract. Surprizingly, of the four immobilized NADP(+) derivatives investigated, only the 8'-azo-linked immobilized NADP(+) derivative proved effective for TBADH affinity purification when used in conjunction with pyrazole (a competitive inhibitor of TBADH activity) as the locking-on ligand. This is in contrast to other NADP(+)-dependent dehydrogenases where the immobilized N(6)-linked cofactor proved to be suitable. While the one-step purification of TBADH to electrophoretic homogeneity is described in the present study (92% yield), results from the model chromatographic studies point to improvements that could be made to the immobilized cofactor derivative to improve its suitability for TBADH bioaffinity purification and to facilitate future large scale protein purification operations. 相似文献
9.
Inositol monophosphatase (IMPase) family of proteins are Mg(2+) activated Li(+) inhibited class of ubiquitous enzymes with promiscuous substrate specificity. Herein, the molecular basis of IMPase substrate specificity is delineated by comparative crystal structural analysis of a Staphylococcal dual specific IMPase/NADP(H) phosphatase (SaIMPase - I) with other IMPases of different substrate compatibility, empowered by in silico docking and Escherichia coli SuhB mutagenesis analysis. Unlike its eubacterial and eukaryotic NADP(H) non-hydrolyzing counterparts, the composite structure of SaIMPase - I active site pocket exhibits high structural resemblance with archaeal NADP(H) hydrolyzing dual specific IMPase/FBPase. The large and shallow SaIMPase - I active site cleft efficiently accommodate large incoming substrates like NADP(H), and therefore, justifies the eminent NADP(H) phosphatase activity of SaIMPase - I. Compared to other NADP(H) non-hydrolyzing IMPases, the profound difference in active site topology as well as the unique NADP(H) recognition capability of SaIMPase - I stems from the differential length and orientation of a distant helix α4 (in human and bovine α5) and its preceding loop. We identified the length of α4 and its preceding loop as the most crucial factor that regulates IMPase substrate specificity by employing a size exclusion mechanism. Hence, in SaIMPase - I, the substrate promiscuity is a gain of function by trimming the length of α4 and its preceding loop, compared to other NADP(H) non-hydrolyzing IMPases. This study thus provides a biochemical - structural framework revealing the length and orientation of α4 and its preceding loop as the predisposing factor for the determination of IMPase substrate specificity. 相似文献
10.
Nozomi Katayama Ken Hayakawa Itaru Urabe Hirosuke Okada 《Enzyme and microbial technology》1984,6(12):538-542
The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD+ oxidoreductase, EC 1.1.1.1 and alcohol:NADP+ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD+ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD+ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD+ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N6-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N6-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9. 相似文献
11.
The oxidation of cytosolic NAD(P)H by external NAD(P)H dehydrogenases in the respiratory chain of plant mitochondria 总被引:2,自引:0,他引:2
Ian M. Møller 《Physiologia plantarum》1997,100(1):85-90
The respiratory chain of plant mitochondria differs from that in mammalian mitochondria by containing several rotenone-insensitive NAD(P)H dehydrogenases. Two of these are located on the outer, cytosolic surface of the inner membrane. One is specific for NADH, the other for NADPH. Only the latter is inhibited by diphenyleneiodonium (DPI). Both of these enzymes are normally dependent upon Ca2+ for activity and this constitutes a potentially important mechanism by which the cell can regulate the oxidation of cytosolic NAD(P)H via the concentration of free Ca2+ . This and other potential regulatory mechanisms such as the substrate concentration and polyamines are discussed. 相似文献
12.
Brenda S. Speer Ludmila Chistoserdova Mary E. Lidstrom 《FEMS microbiology letters》1994,121(3):349-355
Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well. 相似文献
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Antonio de Flora Alessandro Morelli Marco Frascio Giorgio Corte Bruno Curti Monica Galliano Carlotta Gozzer Lorenzo Minchiotti Cristina Mareni Gianfranco Gaetani 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,500(1):109-123
Two sensitive radioimmunoassays, based on a double-antibody technique, were developed which allow detection of nanogram amounts of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and of a so far unknown NADP(H)-binding protein present in human erythrocytes (designated FX).The two proteins isolated in homogeneous form from human erythrocytes were iodinated with 125I by means of lactoperoxidase. Antisera to both purified proteins were raised in rabbits and sequentially adsorbed on human erythrocytes and on human serum before use. No cross-reaction between the two proteins was apparent.Hemolysates from normal as well as from glucose-6-phosphate dehydrogenase-deficient subjects were investigated for their content in both immunoreactive proteins using the two radioimmunoassay methods. This preliminary study showed significantly lowered levels of immunoreactive glucose-6-phosphate dehydrogenase in erythrocytes from subjects carrying the Mediterranean variant of this enzyme (characterized by severe deficiency of catalytic activity), compared with normal subjects. This figure was reversed as concerns the content of immunoreactive FX which was found to be twice as high in glucose-6-phosphate dehydrogenase Mediterranean erythrocytes as in normal ones.The two purified proteins were submitted to a comparative analysis of their chemical properties including NH2-terminal residues, CNBr peptides and tryptic fingerprints. These studies revealed significant differences in the primary structures of the two proteins and therefore tend to exclude FX'x being a discrete product arising from degradation of native glucose-6-phosphate dehydrogenase. Moreover, amino axid analysis and tryptic fingerprints indicated that FX, as well as glucose-6-phosphatase dehydrogenase, is composed of very similar and possibly identical polypeptide chains. 相似文献
15.
Lys601 in NADPH-cytochrome P-450 reductase is modified by reductive alkylation with pyridoxal 5'-phosphate (pyridoxylation). Lys601 is protected against modification by the cosubstrate NADP(H). 相似文献
16.
Hummel W 《Trends in biotechnology》1999,17(12):7023-492
NAD(P)-dependent dehydrogenases are useful catalysts for the synthesis of chiral compounds. Many active and stable enzymes are available that (with high enantioselectivity) reduce ketones or keto acids to chiral alcohols, hydroxy acids or amino acids. For economic reasons, these reactions need coupling to the simultaneous regeneration of NAD(P)H. For preparative applications, three components have to be combined: an appropriate enzyme, an efficient coenzyme-regenerating step and a suitable reaction-engineering technique. 相似文献
17.
A new NADP(H)-dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2-6.4. ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties. ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast. Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast. Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis. 相似文献
18.
Copper ions are known to inactivate a variety of enzymes, and lactate dehydrogenase (LDH) is exceptionally sensitive to the presence of this metal. We now found that NADH strongly enhances the Cu(II)-mediated loss of LDH activity. Surprisingly, NADH was not oxidized in this process and also NAD+ promoted the Cu(II)-dependent inactivation of LDH. Catalase only partly protected the enzyme, whereas hypoxia even enhanced LDH inactivation. NAD(H) accelerated sulfhydryl (SH) group oxidation of LDH by 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and, vice versa, LDH-mediated Cu(II) reduction. LDH activity was preserved by thiol donators and pyruvate and partially preserved by lactate and oxamate. Our results suggest that reactive oxygen species (ROS) are of minor importance for the inactivation of LDH induced by Cu(II)/NADH. We propose that conformational changes of the enzymes' active sites induced by NAD(H)-binding increase the accessibility of active sites' cysteine residues to Cu(II) thereby accelerating their oxidation and, consequently, loss of catalytic activity. 相似文献
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Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called “occluded” intermediary state of the reaction cycle of the intact enzyme. Despite a Kd in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP+ is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2′-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD+ by NADH, indicating that 3-acetylpyridine-NAD+ and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2′-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent. 相似文献