共查询到20条相似文献,搜索用时 15 毫秒
1.
Tiago M. Bandeiras Smilja Todorovic Peter Hildebrandt Manuela M. Pereira Arnulf Kletzin 《BBA》2009,1787(1):37-18098
A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the as-type with reduction potentials of + 200, + 400 and + 160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme as, and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be + 320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane α-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc1 complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc1 complexes of bacteria and mitochondria, however with distinct subunits and heme types. 相似文献
2.
Wang L Liu QM Sung BH An DS Lee HG Kim SG Kim SC Lee ST Im WT 《Journal of biotechnology》2011,156(2):125-133
A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb1, Rb2, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc1 and ginsenoside F2, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent Km and Vmax values of 2.9 ± 0.3 mM and 515.4 ± 38.3 μmol min−1 mg of protein−1 against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb1, Rb2, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc1 and F2 quickly at optimal conditions of pH 5.0 and 37 °C. A little ginsenoside F2 production from ginsenosides Gyp XVII, C-O, and C-Mc1 was observed for the lengthy enzyme reaction caused by the side ability of the enzyme. 相似文献
3.
Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H+ electrochemical gradient (Δμ¯H+, interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Δμ¯H+, like sugar, acts to increase the probability of opening on the periplasmic side of LacY. 相似文献
4.
A new stereoselective preparation of N-aceyl-d-galactosamine (1b) starting from the known p-methoxyphenyl 3,4-O-isopropylidene-6-O-(1-methoxy-1-methylethyl)-β-d-galactopyranoside (10) is described using a simple strategy based on (a) epimerization at C-2 of 10 via oxidation-reduction to give the talo derivative 11, (b) amination with configurational inversion at C-2 of 11 via a SN2-type reaction on its 2-imidazylate, (c) anomeric deprotection of the p-methoxyphenyl β-d-galactosamine glycoside 14, (d) complete deprotection. Applying the same protocol to 2,3:5,6:3′,4′-tri-O-isopropylidene-6′-O-(1-methoxy-1-methylethyl)-lactose dimethyl acetal (4), directly obtained through acetonation of lactose, the disaccharide β-d-GalNAcp-(1→4)-d-Glcp (1a) was obtained with complete stereoselectivity in good (40%) overall yield from lactose. 相似文献
5.
Most commonly used expression systems in bacteria are based on the Escherichia coli lac promoter. Furthermore, lac operon elements are used today in systems and synthetic biology. In the majority of the cases the gratuitous inducers IPTG or TMG are used. Here we report a systematic comparison of lac promoter induction by TMG and IPTG which focuses on the aspects inducer uptake, population heterogeneity and a potential influence of the transacetylase, LacA. We provide induction curves in E. coli LJ110 and in isogenic lacY and lacA mutant strains and we show that both inducers are substrates of the lactose permease at low inducer concentrations but can also enter cells independently of lactose permease if present at higher concentrations. Using a gfp reporter strain we compared TMG and IPTG induction at single cell level and showed that bimodal induction with IPTG occurred at approximately ten-fold lower concentrations than with TMG. Furthermore, we observed that lac operon induction is influenced by the transacetylase, LacA. By comparing two Plac-gfp reporter strains with and without a lacA deletion we could show that in the lacA+ strain the fluorescence level decreased after few hours while the fluorescence further increased in the lacA− strain. The results indicate that through the activity of LacA the IPTG concentration can be reduced below an inducing threshold concentration—an influence that should be considered if low inducer amounts are used. 相似文献
6.
Yoshida H Yamada M Nishitani T Takada G Izumori K Kamitori S 《Journal of molecular biology》2007,374(2):443-453
Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)8 barrel structure, and a metal ion (Mn2+) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn2+, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246. 相似文献
7.
The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α6/α6-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α6/α6-barrel. 相似文献
8.
The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl α-d-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by 1H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl α-d-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 °C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl α-d-galactopyranosiduronic acid) and an α,β-unsaturated aldehyde (methyl 4-deoxy-α-d-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. 1H and 13C NMR data of the products are reported for the α,β-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d6 for all the products for the first time. Oxidation of d-raffinose (α-d-galactopyranosyl-(1-6)-α-d-glucopyranosyl-(1-2)-β-d-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde. 相似文献
9.
Corwin M. Nycholat 《Carbohydrate research》2009,344(12):1397-1411
A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation-reduction sequence at C-2′. The β-manno configurations of the final deprotected congeners 2-7 were confirmed by measurement of 1JC1,H1 heteronuclear and 3J1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the protective epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site. 相似文献
10.
Corwin M. Nycholat 《Carbohydrate research》2009,344(5):555-569
A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation-reduction sequence at C-2′. The β-manno configuration of the final deprotected congeners 2-7 was confirmed by measurement of 1JC1,H1 heteronuclear and 3J1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site. 相似文献
11.
Lactose permease in Escherichia coli (LacY) transports both anomeric states of disaccharides but has greater affinity for α-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of 12 MD simulations of 20-25 ns each with β(α)-d-galactopyranosyl-(1,1)-β-d-galactopyranoside (ββ-(Galp)2) and αβ-(Galp)2 result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu126 and αβ-(Galp)2 has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe118, Asn119, Asn240, His322, Glu325, and Tyr350). Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for αβ-(Galp)2 than ββ-(Galp)2, i.e. −10.7(±0.7) and −3.1(±1.0) kcal/mol, respectively. Of the 12 helices in LacY, helix-IV is the least stable with ββ-(Galp)2 binding resulting in larger distortion than αβ-(Galp)2. 相似文献
12.
From the methanol extract of Cardamine diphylla rhizome, 5′-O-β-d-glucopyranosyl-dihydroascorbigen (1) and 6-hydroxyindole-3-carboxylic acid 6-O-β-d-glucopyranoside (2) were isolated. The structures of the compounds were elucidated using spectroscopic methods. This is the second report on the presence of a glucosylated indole ascorbigen in plants. 相似文献
13.
14.
The aim of this study was to produce rare β-carotene-modified carotenoids possessing 2-O (-H or -glu) and/or 3-O (-H or -glu) functionalities in their β-ionone ring(s) using a recombinant Escherichia coli approach. This involved expressing seven carotenoid biosynthesis genes (crtE, crtB, crtI, crtY, crtZ, crtX and crtG). From the cells of the recombinant E. coli, caloxanthin (β,β-carotene-2,3,2′,3′-tetrol)-3′-β-d-glucose, zeaxanthin (β,β-carotene-3,3′-diol) 3,3′-β-d-diglucoside, and nostoxanthin (β,β-carotene-2,3,3′-triol) (rare carotenoids) were isolated and identified. Caloxanthin 3′-β-d-glucose displayed potent 1O2 quenching activity (IC50 19 μM). 相似文献
15.
Feng Rao Yaning Qi Elavazhagan Murugan Swathi Pasunooti Qiang Ji 《Biochemical and biophysical research communications》2010,398(3):500-505
The recent report of 2′,3′-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2′,3′-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2′,3′-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3′-AMP but not 2′-AMP. The catalytic efficiency (kcat/Km) of each enzyme against 2′,3′-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2′,3′-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3′-AMP is due to the P-O2′ bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2′,3′-cAMP hydrolysis is observed. 相似文献
16.
Bernal-Uruchurtu MI Metta-Magaña AJ Guerrero-Álvarez JA Reyes-Martínez R Tlahuext H 《Carbohydrate research》2008,343(16):2804-2812
The reactivity of N-(2-aminophenyl)-d-glycero-d-gulo-heptonamide (adgha), with the group 12 cations, Zn(II), Cd(II), and Hg(II), was studied in DMSO-d6 solution. The studied system showed a selective coordination to Hg(II), and the products formed were characterized by 1H and 13C NMR in DMSO-d6 solution and fast atom bombardment (FAB+) mass spectra. The expected coordination compounds, [Hg(adgha)](NO3)2 and [Hg(adgha)2](NO3)2, were observed as unstable intermediates that decompose to bis-[2-(d-glycero-d-gulo-hexahydroxyhexyl)-benzimidazole-κN]mercury(II) dinitrate, [Hg(ghbz)2](NO3)2. The chemical transformation of the complexes was followed by NMR experiments, and the nature of the species formed is sustained by a theoretical study done using DFT methodology. From this study, we propose the structure of the complexes formed in solution, the relative stability of the species formed, and the possible role of the solvent in the observed transformations. 相似文献
17.
Raquel da Silva AiresAndrei Stecca Steindorff Marcelo Henrique Soller RamadaSaulo José Linhares de Siqueira Cirano José Ulhoa 《Carbohydrate polymers》2012,87(2):1219-1223
Trichoderma asperellum produces two extracellular 1,3-β-d-glucanase upon induction with cell walls from Rhizoctonia solani. A minor 1,3-β-d-glucanase was purified to homogeneity by ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. A typical procedure provided 13.8-fold purification with 70% yield. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 27 kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45 °C. It was thermostable at 40 °C, and retained 75% activity after 60 min at 45 °C. The Km and Vmax values for 1,3-β-d-glucanase, using laminarin as substrate, were 0.323 mg ml−1 and 0.315 U min−1, respectively. The enzyme was strongly inhibited by Hg2+ and SDS. The enzyme was only active toward glucans containing β-1,3-linkages. Peptide sequences showed similarity with two endo-1,3(4)-β-d-glucanases from Aspergillus fumigatus Af293when compared against GenBank non-redundant database. 相似文献
18.
The lactose (lac) repressor is an allosteric protein that can respond to environmental changes. Mutations introduced into the DNA binding domain and the effector binding pocket affect the repressor's ability to respond to its environment. We have demonstrated how the observed phenotype is a consequence of altering the thermodynamic equilibrium constants. We discuss mutant repressors, which (1) show tighter repression; (2) induce with a previously noninducing species, orthonitrophenyl-β-d-galactoside; and (3) transform an inducible switch to one that is corepressed. The ability of point mutations to change multiple thermodynamic constants, and hence drastically alter the repressor's phenotype, shows how allosteric proteins can perform a wide array of similar yet distinct functions such as that exhibited in the Lac/Gal family of bacterial repressors. 相似文献
19.
Masayuki Okuyama Hironori Hondoh Haruhide Mori Isao Tanaka 《Journal of molecular biology》2009,392(5):1232-312
Glycoside hydrolase family 97 (GH 97) is a unique glycoside family that contains inverting and retaining glycosidases. Of these, BtGH97a (SusB) and BtGH97b (UniProtKB/TrEMBL entry Q8A6L0), derived from Bacteroides thetaiotaomicron, have been characterized as an inverting α-glucoside hydrolase and a retaining α-galactosidase, respectively. Previous studies on the three-dimensional structures of BtGH97a and site-directed mutagenesis indicated that Glu532 acts as an acid catalyst and that Glu439 and Glu508 function as the catalytic base in the inverting mechanism. However, BtGH97b lacks base catalysts but possesses a putative catalytic nucleophilic residue, Asp415. Here, we report that Asp415 in BtGH97b is the nucleophilic catalyst based on the results of crystal structure analysis and site-directed mutagenesis study. Structural comparison between BtGH97b and BtGH97a indicated that OD1 of Asp415 in BtGH97b is located at a position spatially identical with the catalytic water molecule of BtGH97a, which attacks on the anomeric carbon from the β-face (i.e., Asp415 is poised for nucleophilic attack on the anomeric carbon). Site-directed mutagenesis of Asp415 leads to inactivation of the enzyme, and the activity is rescued by an external nucleophilic azide ion. That is, Asp415 functions as a nucleophilic catalyst. The multiple amino acid sequence alignment of GH 97 members indicated that almost half of the GH 97 enzymes possess base catalyst residues at the end of β-strands 3 and 5, while the other half of the family show a conserved nucleophilic residue at the end of β-strand 4. The different positions of functional groups on the β-face of the substrate, which seem to be due to “hopping of the functional group” during evolution, have led to divergence of catalytic mechanism within the same family. 相似文献
20.
The transformation of (5R)-2,6-di-O-benzyl-5-C-methoxy-β-d-galactopyranosyl-(1→4)-2,3:5,6-di-O-isopropylidene-aldehydo-d-glucose dimethyl acetal (8) into partially protected derivatives of d-xylo- and l-lyxo-aldohexos-5-ulose has been reported, applying appropriate epimerisation methods to its 3′-O- and 4′-O-protected alcoholic derivatives. 相似文献