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1.
Anu Nutt Andres Salumets Gunnar Henriksson Veljo Sild Gunnar Johansson 《Biotechnology letters》1997,19(4):379-384
Cellobiose dehydrogenase from Phanerochaete chrysosporium produces HO by electron transfer between cellobiose and O with a lower yield than the 1:1:1 molar ratio displayed by Aspergillus niger. glucose oxidase in the similar reaction between glucose and O. The discrepancy could best be explained if both a FentonÕs reaction and the spontaneous reactivity of the oxygen species formed were taken into account. 相似文献
2.
Summary Purified plasma membrane vesicles isolated both by aqueous twoPhase methods and by free-flow electrophoresis from homogenates prepared in the presence of 10 mM ascorbate, oxidized external NADH at rates of about 15 nanomoles/min/mg protein. The rate in the isolated vesicles was accelerated, without perceptible lag, 1.5-to 2-fold by 1 to 10 M auxin (2,4-dichlorophenoxyacetic acid or indole-3-acetic acid). The reaction would be expected to result in acidification of the vesicle interiors and is proposed as a mechanism to account for auxin-induced acidification of cytoplasmin vitro. 相似文献
3.
I. S. Severina V. I. Fedchenko A. V. Veselovsky A. E. Medvedev 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2016,10(2):97-109
Renalase is a recently discovered secretory protein, which plays a certain (still poorly understood) role in regulation of blood pressure. The review summarizes own and literature data on structure and catalytic properties of renalase accumulated since the first publication on this protein (2005). Initial reports on FADdependent amine oxidase activity were not confirmed in independent experiments performed in different laboratories. In addition, proposed amine oxidase activity of circulating extracellular renalase requires the presence of FAD, which has not been detected either in blood or urinary renalase. Moreover, renalase excreted into urine lacks its N-terminal peptide, which is ultimately needed for accommodation of the FAD cofactor. Results of the Aliverti’s group on NAD(P)H binding by renalase and weak diaphorase activity of this enzyme stimulated further studies of renalase as NAD(P)H oxidase catalyzing reaction of catecholamine co-oxidation. However, physiological importance of such extracellular catecholamine-metabolizing activity remains unclear due to existence of much more active enzymatic systems (e.g., neutrophil NAD(P)H oxidase, xanthine oxidase/xanthine) in circulation, which can perform such co-oxidation reactions. Recently α-NAD(P)H oxidase/anomerase activity of renalase, which also promotes oxidative conversion of β-NADH isomers inhibiting activity of NAD-dependent dehydrogenases, has been described. However, its possible contribution to the antihypertensive effect of renalase remains unclear. Thus, the antihypertensive effect of renalase still remains a phenomenon with unclear biochemical mechanim(s) and functions of intracellular and extracellular (circulating) renalases obviously differ. 相似文献
4.
Na Nakorn P. Suphantharika M. Udomsopagit S. Surareungchai W. 《World journal of microbiology & biotechnology》2003,19(5):479-485
An amperometric L-glutamate oxidase (GLOD) electrode based on a multilayer of polymer films was developed for the high selective determination of L-glutamate. The multilayer film consisted of three layers as the following configuration i.e., inner membrane of electropolymerized 1,3-diaminobenzene, middle layer of L-GLOD entrapped in photopolymerized poly(vinylferrocene)–poly(ethylene glycol) hydrogel polymer, and outer dialysis membrane. In this manner, the sensor could eliminate interferences and was able to work at a low potential poised at +0.085 V vs. Ag/AgCl. When used in a flow injection system, the sensor responded to L-glutamate in the range 0.5–8.0 mM, with a sensitivity of 9.48 nA mM–1. The sensor was stable for 5 days of continuous uses (250 assays) and retained 60% activity after 16 days. When used to analyse the L-glutamate contents of a number of different off-the-shelf soy sauces, the sensor gave results in good agreement with the standard colorimetric method. 相似文献
5.
Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high (a(H)) or low (a(L)) midpoint potential (E(m)) by comparing their occupancy during redox titrations carried out when the membrane potential (ΔΨ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a(L) has a maximum at 605nm whereas the spectrum of a(H) has a maximum at 602nm. The ΔΨ-dependent shift in the E(m) of a(H) was too great to be accounted for by electron transfer from cytochrome c to heme a against ΔΨ but was consistent with a model in which a(H) is formed after proton uptake against ΔΨ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E(m) changes during redox titrations. The redox anti-cooperativity between heme a and heme a(3), and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a(3) with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes. 相似文献
6.
Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 (‘copper signal’) of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (?170 to ?190°C). For some sets of samples spectra were recorded in the range 500–1100 nm. A particular effort was made to study this correlation with what are called ‘mixed valence’ states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205–215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10–15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700–800 nm region did not appear to be more useful than the 800–900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme. 相似文献
7.
Gulden Koclar Avci Nursen Coruh Ufuk Bolukbasi Zumrut B. Ogel 《Applied microbiology and biotechnology》2013,97(2):661-672
The thermophilic fungus Scytalidium thermophilum produces a novel bifunctional catalase with an additional phenol oxidase activity (CATPO); however, its phenol oxidation spectrum is not known. Here, 14 phenolic compounds were selected as substrates, among which (+)-catechin, catechol, caffeic acid, and chlorogenic acid yielded distinct oxidation products examined by reversed-phase HPLC chromatography method. Characterization of the products by LC-ESI/MS and UV–vis spectroscopy suggests the formation of dimers of dehydrocatechin type B (hydrophilic) and type A (hydrophobic), as well as oligomers, namely, a trimer and tetramer from (+)-catechin, the formation of a dimer and oligomer of catechol, a dimer from caffeic acid with a caffeicin-like structure, as well as trimeric and tetrameric derivatives, and a single major product from chlorogenic acid suggested to be a dimer. Based on the results, CATPO oxidizes phenolic compounds ranging from simple phenols to polyphenols but all having an ortho-diphenolic structure in common. The enzyme also appears to have stereoselectivity due to the oxidation of (+)-catechin, but not that of epicatechin. It is suggested that CATPO may contribute to the antioxidant mechanism of the fungus and may be of value for future food and biotechnology applications where such a bifunctional activity would be desirable. 相似文献
8.
C I Levene M P O'Shea M J Carrington 《The International journal of biochemistry》1988,20(12):1451-1456
1. Treatment of chick embryos with two lathyrogens lowered lysyl oxidase and increased collagen extractability. 2. Subsequent treatment with pyridoxal restored both parameters towards normal, whereas PQQ treatment was less effective. 3. These results suggest the requirement of a pyridoxal derivative for the formation of the enzyme, acting either as cofactor or because its formation requires some pyridoxal-dependent enzyme. The cochromatography of the enzyme with [3H]pyridoxine-derived radioactivity supports the cofactor role. 4. The conclusions of other authors that lysyl oxidase contains PQQ relates to enzymes from other species or to amine oxidases not characterised as lysyl oxidase. 相似文献
9.
《Insect Biochemistry》1978,8(4):231-236
Monoamine oxidase (MAO) activity was demonstrated in intact brains of larvae from the European corn borer, Ostrinia nubilalis using 2-(2′-benzothiazolyl)-5-stryl-3-(4′-phthalhydrazidyl) tetrazolium chloride (BSPT). With tryptamine as the substrate, MAO specific activity was restricted to mitochondria within perineurial cells. The basic BSPT methodology was modified by the substitution of 2% dimethylsulfoxide for dimethylformamide in the incubation medium. This yielded increased permeability of the brain to the incubation medium, presumably by disrupting the integrity of the blood-brain barrier. A discussion of possible reasons for the previous inability to demonstrate insect neuronal MAO activity is presented. 相似文献
10.
Haruhito Tsuge Yasumasa Kuroda Atsuhiro Iwamoto Kazuji Ohashi 《Archives of biochemistry and biophysics》1982,217(2):479-484
Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes. 相似文献
11.
《Bioorganic & medicinal chemistry letters》2014,24(12):2758-2763
In the present study, a series of fifteen α-tetralone (3,4-dihydro-2H-naphthalen-1-one) derivatives were synthesised and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The α-tetralone derivatives examined are structurally related to a series of chromone (1-benzopyran-4-one) derivatives which has previously been shown to act as MAO-B inhibitors. The results document that the α-tetralones are highly potent MAO-B inhibitors with all compounds exhibiting IC50 values in the nanomolar range (<78 nM). Although most compounds are selective inhibitors of MAO-B, the α-tetralones are also potent MAO-A inhibitors with ten compounds exhibiting IC50 values in the nanomolar range (<792 nM). The most potent MAO-B inhibitor, 6-(3-iodobenzyloxy)-3,4-dihydro-2H-naphthalen-1-one, exhibits an IC50 value of 4.5 nM with a 287-fold selectivity for MAO-B over the MAO-A isoform, while the most potent MAO-A inhibitor, 6-(3-cyanobenzyloxy)-3,4-dihydro-2H-naphthalen-1-one, exhibits an IC50 value of 24 nM with a 3.25-fold selectivity for MAO-A. Analyses of the structure–activity relationships for MAO inhibition show that substitution on the C6 position of the α-tetralone moiety is a requirement for MAO-A and MAO-B inhibition, and that a benzyloxy substituent on this position is more favourable for MAO-A inhibition than phenylethoxy and phenylpropoxy substitution. For MAO-B inhibition, alkyl and halogen substituents on the meta and para positions of the benzyloxy ring enhance inhibitory potency. It may be concluded that α-tetralone derivatives are promising leads for design of therapies for Parkinson’s disease and depression. 相似文献
12.
Sandra Scheele Dan Oertel Johannes Bongaerts Stefan Evers Hendrik Hellmuth Karl-Heinz Maurer Michael Bott Roland Freudl 《Microbial biotechnology》2013,6(2):202-206
Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol–xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies.The secretory expression of recombinant proteins can offer significant process advantages over cytosolic production strategies, since secretion into the growth medium greatly facilitates downstream processing and therefore can significantly reduce the costs of producing a desired target protein (Quax, 1997). And, in fact, the enormous secretion capacity of certain Gram-positive bacteria (e.g. various Bacillus species) has been used since many years in industry for the production of mainly host-derived secretory proteins such as proteases and amylases, resulting in amounts of more than 20 g l−1 culture medium (Harwood and Cranenburg, 2008). In contrast, attempts to use Bacillus species for the secretory production of heterologous proteins have often failed or led to disappointing results, a fact that, among other reasons, could in many cases be attributed to the presence of multiple cell wall-associated and secreted proteases that rapidly degraded the heterologous target proteins (Li et al., 2004; Sarvas et al., 2004; Westers et al., 2011). Therefore, an increasing need exists to explore alternative host systems with respect to their ability to express and secrete problematic and/or complex heterologous proteins of biotechnological interest.So far, the Gram-positive bacterium Corynebacterium glutamicum has been used in industry mainly for the production of amino acids and other low-molecular weight compounds (Leuchtenberger et al., 2005; Becker and Wittmann, 2011; Litsanov et al., 2012). However, various recent reports have indicated that C. glutamicum might likewise possess a great potential as an alternative host system for the secretory expression of foreign proteins. Corynebacterium glutamicum belongs to a class of diderm Gram-positive bacteria that, besides the cytoplasmic membrane, possess an additional mycolic acid-containing outer membrane-like structure that acts as an extremely efficient permeability barrier for hydrophilic compounds (Hoffmann et al., 2008; Zuber et al., 2008). Despite this fact, an efficient secretion of various heterologous proteins into the growth medium of this microorganism has been observed (e.g. Billman-Jacobe et al., 1995; Meissner et al., 2007; Kikuchi et al., 2009; Tateno et al., 2009; Tsuchidate et al., 2011).In bacteria, two major export pathways exist for the transport of proteins across the cytoplasmic membrane that fundamentally differ with respect to the folding status of their respective substrate proteins during the actual translocation step. The general secretion (Sec) system transports its substrates in a more or less unfolded state and folding takes places on the trans side of the membrane after the actual transport event (Yuan et al., 2010; du Plessis et al., 2011). In contrast, the alternative twin-arginine translocation (Tat) system translocates its substrates in a fully folded form and therefore provides an attractive alternative for the secretory production of proteins that cannot be produced in a functional form via the Sec route (Brüser, 2007). Carbohydrate oxidases are biotechnologically interesting enzymes (van Hellemond et al., 2006) that are excluded from Sec-dependent secretion since they depend on a tightly or covalently bound cofactor for their activity and, for this reason, require that their folding and cofactor insertion has to take place in the cytosol. Because C. glutamicum has shown to be an excellent host for the Tat-dependent secretion of the cofactor-less model protein GFP (Meissner et al., 2007; Teramoto et al., 2011), we now asked whether it is likewise possible to secrete a cofactor-containing enzyme into the supernatant of C. glutamicum using the same protein export route.As a model protein, we chose the sorbitol–xylitol oxidase (SoXy) from Streptomyces coelicolor, a normally cytosolic enzyme that possesses a covalently bound FAD molecule as cofactor (Heuts et al., 2007; Forneris et al., 2008). FAD is incorporated into the apoprotein in a post-translational and self-catalytic process that only occurs if the polypeptide chain has adopted a correctly folded structure (Heuts et al., 2007; 2009). To direct SoXy into the Tat export pathway of C. glutamicum, we constructed a gene encoding a TorA–SoXy hybrid precursor in which SoXy is fused to the strictly Tat-specific signal peptide of the periplasmic Escherichia coli Tat substrate trimethylamine N-oxide reductase (TorA) (Fig. 1) which, in our previous study, has been proven to be a functional and strictly Tat-specific signal peptide also in C. glutamicum (Meissner et al., 2007). The corresponding torA–soxy gene was cloned into the expression vector pEKEx2 (Eikmanns et al., 1991) under the control of an IPTG-inducible Ptac promotor. After transformation of the resulting plasmid pTorA–SoXy into the C. glutamicum ATCC13032 wild-type strain, two independent colonies of the resulting recombinant C. glutamicum (pTorA–SoXy) strain and, as a control, a colony of a strain that contained the empty expression vector without insert [C. glutamicum (pEKEx2)] were grown in CGXII medium (Keilhauer et al., 1993) at 30°C for 16 h in the presence of 1 mM IPTG. Subsequently, the proteins present in the culture supernatants were analysed by SDS-PAGE followed by staining with Coomassie blue. As shown in Fig. 2, in the supernatants of the pTorA–SoXy-containing cells (lanes 3 and 4), a prominent protein band of approximately 44 kDa can be detected, the size of which is very similar to the calculated molecular mass (44.4 kDa) of SoXy. Since this band is completely lacking in the supernatant of the control strain (lane 2), this strongly suggests that this band corresponds to SoXy that has been secreted into the culture supernatant of C. glutamicum (pTorA–SoXy). And, in fact, this suggestion was subsequently confirmed in a direct way by MALDI-TOF mass spectrometry after extraction of the protein out of the gel followed by tryptic digestion (Schaffer et al., 2001) (data not shown).Open in a separate windowFigure 1The TorA–SoXy hybrid precursor protein. Upper part: Schematic drawing of the relevant part of the pTorA–SoXy expression vector. Ptac, IPTG-inducible tac promotor. RBS, ribosome binding site. To maintain the authentic TorA signal peptidase cleavage site, the first four amino acids of the mature TorA protein (black bar) were retained in the TorA–SoXy fusion protein. White bar: TorA signal peptide (TorASP); grey bar: SoXy (amino acids 2–418). Lower part: Amino acid sequence of the signal peptide and early mature region of the TorA–SoXy hybrid precursor. The twin-arginine consensus motif of the TorA signal peptide is underlined. The four amino acids derived from mature TorA are shown in italics. The signal peptidase cleavage site is indicated by an arrowhead.Open in a separate windowFigure 2Secretion of SoXy into the growth medium of C. glutamicum. Cells of C. glutamicum ATCC13032 containing the empty vector pEKEx2 and two independently transformed colonies of C. glutamicum (pTorA–SoXy) were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with CGXII medium (Keilhauer et al., 1993) and inoculated to an OD600 of 0.5 in 5 ml of fresh CGXII medium containing 1 mM IPTG. After 16 h of further growth at 30°C, the supernatant fractions were prepared as described previously (Meissner et al., 2007). Samples corresponding to an equal number of cells were subjected to SDS-PAGE followed by staining with Coomassie blue. Lane 1, molecular mass marker (kDa). Lane 2, C. glutamicum (pEKEx2); lanes 3 and 4, C. glutamicum (pTorA–SoXy). The position of the secreted SoXy protein is indicated by an arrow.Next, the supernatant of C. glutamicum (pTorA–SoXy) was analysed for SoXy enzyme activity by measuring the production of H2O2 that is formed during the enzymatic conversion of sorbitol to fructose (Meiattini, 1983). Six hours after induction of gene expression by 1 mM IPTG, an enzymatic activity of 10.3 ± 1.6 nmol min−1 ml−1 could be determined in the supernatant of C. glutamicum (pTorA–SoXy). In contrast, no such activity was found in the supernatant of the control strain C. glutamicum (pEKEx2). From these results we conclude that we have succeeded in the secretion of enzymatically active and therefore FAD cofactor-containing SoXy into the culture supernatant of C. glutamicum.Finally, we examined whether the secretion of SoXy had in fact occurred via the Tat pathway of C. glutamicum. Plasmid pTorA–SoXy was used to transform C. glutamcium ATCC13032 wild type and a C. glutamicum ΔTatAC mutant strain that lacks two essential components of the Tat transport machinery and therefore does not possess a functional Tat translocase (Meissner et al., 2007). The corresponding cells were grown in BHI medium (Difco) at 30°C in the presence of 1 mM IPTG for 6 h. Subsequently, the proteins present in the cellular and the supernatant fractions of the corresponding cells were analysed by SDS-PAGE followed by Western blotting using SoXy-specific antibodies. As shown in Fig. 3, polypeptides corresponding to the unprocessed TorA–SoXy precursor and some minor smaller degradation products of it can be detected in the cellular fractions of both the wild-type and the ΔTatAC deletion strains (lanes 3 and 5). In the supernatant fraction of the Tat+ wild-type strain (lane 4), but not that of the ΔTatAC strain (lane 6), a polypeptide corresponding to mature SoXy is present, clearly showing that export of SoXy in the wild-type strain had occurred in a strictly Tat-dependent manner. Another noteworthy finding is the observation that hardly any mature SoXy protein accumulated in the cellular fraction of the Tat+ wild-type strain (lane 3), indicating that SoXy is, after its Tat-dependent translocation across the cytoplasmic membrane and processing by signal peptidase, rapidly transported out of the intermembrane space across the mycolic acid-containing outer membrane into the supernatant. However, the mechanism of how proteins cross this additional permeability barrier is completely unknown so far (Bitter et al., 2009).Open in a separate windowFigure 3Transport of TorA–SoXy occurs in a strictly Tat-dependent manner. Plasmid pTorA–SoXy was transformed into C. glutamcium ATCC13032 (Tat+) and a C. glutamicum ΔTatAC mutant that lacks a functional Tat translocase (Meissner et al., 2007). As a control, the empty pEKEx2 expression vector was transformed into C. glutamicum ATCC13032 (Tat+). The respective strains were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with BHI and resuspended in 20 ml of fresh BHI medium containing 1 mM IPTG. After 6 h of further growth at 30°C, the cellular (C) and supernatant (S) fractions were prepared as described previously (Meissner et al., 2007). Samples of the C and S fractions were subjected to SDS-PAGE followed by immunoblotting using anti-SoXy antibodies as indicated at the top of the figure. Lanes 1 and 2: C. glutamicum ATCC13032 (pEKEx2); lanes 3 and 4: C. glutamicum ATCC13032 (pTorA–SoXy); lanes 5 and 6: C. glutamicum ΔTatAC (pTorA–SoXy). Asterisk: TorA–SoXy precursor; arrow: secreted SoXy protein. The positions of molecular mass markers (kDa) are indicated at the left margin of the figure.To the best of our knowledge, our results represent the first documented example of the successful secretion of a normally cytosolic, cofactor-containing protein via the Tat pathway in an active form into the culture supernatant of a recombinant expression host. Our results clearly show that, also for this biotechnologically very interesting class of proteins, a secretory production strategy can be a promising alternative to conventional intracellular expression strategies. Besides for SoXy and other FAD-containing carbohydrate oxidases, for which various applications are perceived by industry such as the in situ generation of hydrogen peroxide for bleaching and disinfection performance in technical applications, their use in the food and drink industry, as well as their use in diagnostic applications and carbohydrate biosynthesis processes (Oda and Hiraga, 1998; Murooka and Yamashita, 2001; van Hellemond et al., 2006; Heuts et al., 2007), a secretory production strategy might now be an attractive option also for biotechnologically relevant enzymes that are used as biocatalysts in chemo-enzymatic syntheses and that possess cofactors other than FAD, such as pyridoxal-5′-phosphate (PLP)-dependent ω-transaminases (Mathew and Yun, 2012) or various thiamin diphosphate (TDP)-dependent enzymes (Müller et al., 2009). 相似文献
13.
Ahsana Dar Khalid M. Khan Humayun S. Ateeq Shagufta Khan Shagufta Rahat Shahnaz Perveen 《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):269-274
A series of hydrazine derivatives was synthesized in order to evaluate their monoamine oxidase A (MAO-A) inhibitory effects. MAO-A inhibitory activity of 4-tosyl benzoic acid carbohydrazide was quite potent, similarly to that of the corresponding 4-benzyloxy-benzoic acid carbohydrazide and its N-cyanoethylated derivative. Structural variations of these compounds, such as the replacement of the 4-substitutent, of the aromatic ring on which the carbohydrazide moiety is grafted, as well as cyclization of the hydrazide moiety in five- or six-membered rings caused either significant decline or complete loss of MAO inhibitory properties. The most active compound (4-tosyl benzoic acid carbohydrazide) was also subjected to the forced swim test, an animal model of depression, eliciting a marked reduction in immobility time in rats, without affecting the locomotor activity, implying that it possesses anti-depressant properties due to inhibition of MAO type-A. 相似文献
14.
The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of and H2O2 during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries. 相似文献
15.
Nag S Lehmann L Kettschau G Heinrich T Thiele A Varrone A Gulyas B Halldin C 《Bioorganic & medicinal chemistry》2012,20(9):3065-3071
The aim of this study was to synthesize and evaluate a novel fluorine-18 labeled analogue of rasagiline (6) as a PET radioligand for monoamine oxidase B (MAO-B). The corresponding non-radioactive fluorine-19 ligand, (1S,2S)-2-fluoro-N-(prop-2-yn-1-yl)indan-1-amine (4), was characterized in in vitro assays. The precursor compound (3aS,8aR)-3-(prop-2-yn-1-yl)-3,3a,8,8a-tetrahydroindeno[1,2-d][1,2,3]oxathiazole 2,2-dioxide (3) and reference standard 4 were synthesized in multi-step syntheses. Recombinant human MAO-B and MAO-A enzyme preparations were used in order to determine IC(50) values for compound 4 by use of an enzymatic assay employing kynuramine as substrate. Radiolabeling was accomplished by a two-step synthesis, compromising a nucleophilic substitution followed by hydrolysis of the sulphamidate group. Human whole hemisphere autoradiography (ARG) was performed with [(18)F]fluororasagiline. Blocking experiments with pirlindole (MAO-A), L-deprenyl and rasagiline (MAO-B) were conducted to demonstrate the specificity of the binding. A positron emission tomography (PET) study was carried out in a cynomolgus monkey where time activity curves for whole brain and regions with high and low MAO-B activity were recorded. Radiometabolites were measured in monkey plasma using gradient HPLC. Compound 4 inhibited MAO-B with an IC(50) of 27 nM and MAO-A with an IC(50) of 2.3 μM. Radiolabeling of precursor 3 and subsequent hydrolysis of the protecting group towards (1S,2S)-2-[(18)F]fluoro-N-(prop-2-yn-1-yl)indan-1-amine (6) was successfully accomplished with an radiochemical yield of 40-70%, a radiochemical purity higher than 99% and a specific radioactivity higher than 200GBq/μmol. ARG demonstrated selective binding for [(18)F]fluororasagiline (6) to MAO-B containing brain regions, for example, striatum. The initial uptake in the monkey brain was 250% SUV at 4 min post injection. The highest amounts of radioactivity were observed in the striatum and thalamus as expected whereas in the cortex and cerebellum lower levels were observed. Metabolite studies demonstrated 30% unchanged radioligand at 90 min post injection. Our investigations demonstrated that the new ligand [(18)F]fluororasagiline (6) binds specifically to MAO-B in vitro and has a MAO-B specific binding pattern in vivo. Thus, it could serve as a novel potential candidate for human PET studies. 相似文献
16.
The crystal structure of glycolate oxidase from spinach has been determined to 5.5 Å resolution, using two isomorphous heavy-atom derivatives and their anomalous contributions. In the electron density map the boundaries of the octameric molecules are clearly seen. The subunit molecular weight is 37,000. Two protomers are in very close contact around one of the crystallographic 2-fold axes. Four such dimers are in contact around the 4-fold axis, so that the glycolate oxidase molecules are arranged as octamers with 422 symmetry in the crystal lattice. The roughly spherical octameric molecules have a diameter of approximately 100 Å. These octamers are arranged in a network, such that large solvent channels, approximately 60Å in diameter, pass right through the crystal lattice.The secondary structure of two-thirds of the subunit density has been interpreted in terms of eight consecutive β strand-α-helix units forming a cylinder very similar to the structure of triose phosphate isomerase. This interpretation is based on the very characteristic arrangement of the eight helices which form such a cylinder. The binding site of a substrate analogue, thioglycolate, has been localized in a deep cleft of the subunit at one end of the βα-barrel close to its axis. 相似文献
17.
Franziska Wendt Christian Näther Felix Tuczek 《Journal of biological inorganic chemistry》2016,21(5-6):777-792
Four new imidazole-based ligands, 4-((1H-imidazol-4-yl)methyl)-2-phenyl-4,5-dihydrooxyzole (L OL 1), 4-((1H-imidazol-4-yl)methyl)-2-(tert-butyl)-4,5-dihydrooxyzole (L OL 2), 4-((1H-imidazol-4-yl)methyl)-2-methyl-4,5-dihydrooxyzole (L OL 3), and N-(2,2-dimethylpropylidene)-2-(1-trityl-1H-imidazol-4-yl-)ethyl amine (L imz 1), have been synthesized. The corresponding copper(I) complexes [Cu(I)(L OL 1)(CH3CN)]PF6 (CuL OL 1), [Cu(I)(L OL 2)(CH3CN)]PF6 (CuL OL 2), [Cu(I)(L OL 3)(CH3CN)]PF6 (CuL OL 3), [Cu(I)(L imz 1)(CH3CN)2]PF6 (CuL imz 1) as well as the Cu(I) complex derived from the known ligand bis(1-methylimidazol-2-yl)methane (BIMZ), [Cu(I)(BIMZ)(CH3CN)]PF6 (CuBIMZ), are screened as catalysts for the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC-H2) to 3,5-di-tert-butylquinone (3,5-DTBQ). The primary reaction product of these oxidations is 3,5-di-tert-butylsemiquinone (3,5-DTBSQ) which slowly converts to 3,5-DTBQ. Saturation kinetic studies reveal a trend of catalytic activity in the order CuL OL 3 ≈ CuL OL 1 > CuBIMZ > CuL OL 2 > CuL imz 1. Additionally, the catalytic activity of the copper(I) complexes towards the oxygenation of monophenols is investigated. As substrates 2,4-di-tert-butylphenol (2,4-DTBP-H), 3-tert-butylphenol (3-TBP-H), 4-methoxyphenol (4-MeOP-H), N-acetyl-l-tyrosine ethyl ester monohydrate (NATEE) and 8-hydroxyquinoline are employed. The oxygenation products are identified and characterized with the help of UV/Vis and NMR spectroscopy, mass spectrometry, and fluorescence measurements. Whereas the copper complexes with ligands containing combinations of imidazole and imine functions or two imidazole units (CuL imz 1 and CuBIMZ) are found to exhibit catalytic tyrosinase activity, the systems with ligands containing oxazoline just mediate a stoichiometric conversion. Correlations between the structures of the complexes and their reactivities are discussed. 相似文献
18.
Fructosamine oxidases (FAOXs) are flavin-containing enzymes that catalyze the oxidative deglycation of low molecular weight fructosamines or Amadori products. The fructosamine substrate is oxidized by the flavin in the reductive half-reaction, and the reduced flavin is then oxidized by molecular oxygen in the oxidative half-reaction. The crystal structure of FAOX-II from Aspergillus fumigatus reveals a unique interaction between Lys53 and the isoalloxazine. The ammonium nitrogen of the lysine is in contact with and nearly centered over the aromatic ring of the flavin on the si-face. Here, we investigate the importance of this unique interaction on the reactions catalyzed by FAOX by studying both half-reactions of the wild-type and Lys53 mutant enzymes. The positive charge of Lys53 is critical for flavin reduction but plays very little role in the reaction with molecular oxygen. The conservative mutation of Lys53 to arginine had minor effects on catalysis. However, removing the charge by replacing Lys53 with methionine caused more than a million-fold decrease in flavin reduction, while only slowing the oxygen reaction by ~30-fold. 相似文献
19.
A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent Km value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay. 相似文献
20.
Wu X Kobori H Orita I Zhang C Imanaka T Xing XH Fukui T 《Biotechnology and bioengineering》2012,109(1):53-62
A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkNOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of TkNOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP(+) and NAD(+) in alcohol dehydrogenase (ADH)-catalyzed enantioselective oxidation of racemic 1-phenylethanol. NADP(+) regeneration at 30°C was performed by TkNOX coupled with (R)-specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)-1-phenylethanol. The use of TkNOX with moderately thermostable (S)-specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD(+) regeneration at high temperatures. Optically pure (R)-1-phenylethanol was successfully obtained by this system after a shorter reaction time at 45-60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of TkNOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air. 相似文献