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1.
Ségolène Colrat Alain Latché Monique Guis Jean-Claude Pech Mondher Bouzayen Jean Fallot Jean-Paul Roustan 《Plant physiology》1999,119(2):621-626
Eutypine
(4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin
produced by Eutypa lata, the causal agent of eutypa
dieback in the grapevine (Vitis vinifera). Eutypine is
enzymatically converted by numerous plant tissues into eutypinol
(4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite
that is nontoxic to grapevine. We report a four-step procedure for the
purification to apparent electrophoretic homogeneity of a
eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna
radiata) hypocotyls. The purified protein is a monomer of 36
kD, uses NADPH as a cofactor, and exhibits a
Km value of 6.3 μm for
eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The
enzyme failed to catalyze the reverse reaction using eutypinol as a
substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2
to 7.5. These data strongly suggest that ERE is an aldehyde reductase
that could probably be classified into the aldo-keto reductase
superfamily. We discuss the possible role of this enzyme in eutypine
detoxification.Many pathogenic bacteria and fungi produce toxins that interfere
with various functions of plant cells and may affect plant defense
mechanisms (Durbin, 1981). Toxin production is commonly associated with
disease severity and can be involved in colonization or systemic
invasion by the pathogen (Schäfer, 1994). Toxin resistance has
been shown in most cases to be based on the ability of the plant to
metabolically detoxify pathogen toxins (Meeley and Walton, 1991; Zhang
and Birch, 1997; Zweimuller et al., 1997). Few cloned toxin-resistance
genes that encode proteins involved in detoxification mechanisms have
been described (Utsumi et al., 1988; Johal and Briggs, 1992; Zhang and
Birch, 1997). In many cases a relationship exists between toxin
tolerance and resistance to the disease (Anzai et al., 1989; Meeley et
al., 1992). The availability of toxin-resistance genes will permit a
greater understanding of the mechanisms causing plant disease and will
also set the stage for engineering resistance to plant disease (Keen,
1993).Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a
toxin produced by the ascomycete fungus Eutypa lata (Pers.:
Fr.) Tul., the causal agent of eutypa dieback (Tey-Rulh et al., 1991).
This disease is responsible for considerable loss in yield and is the
most devastating disease of grapevine (Vitis vinifera) in
many countries (Moller and Kasamitis, 1981; Munkvold et al., 1994). The
fungus infects the stock through pruning wounds and is present in the
xylem and phloem of the vine trunk and branches (Moller and
Kasamitis, 1978; Duthie et al., 1991). After a long incubation period,
a canker forms around the infected wound. The toxin synthesized by the
fungus in the trunk is believed to be transported by the sap to the
herbaceous parts of the vine (Fallot et al., 1997). Eutypine penetrates
grapevine cells through passive diffusion and its accumulation in the
cytoplasm has been explained by an ion-trapping mechanism related to
the ionization state of the molecule (Deswarte et al., 1996b). In the
cell the effects of eutypine include reduction of adenylated nucleotide
content, inhibition of succinate dehydrogenase, uncoupling of oxidative
phosphorylation, and mitochondrial swelling (Deswarte et al., 1996a).Symptoms of eutypa dieback in the herbaceous part of the plant lead to
dwarfed and withered new growth of branches, marginal necrosis of the
leaves, dryness of the inflorescence, and, finally, death of one or
more branches (Moller and Kasamitis, 1981). The toxin appears to be an
important virulence factor involved in symptom development of the
disease (Deswarte et al., 1996a). However, the absence of
toxin-deficient mutants of the fungus and its long incubation period in
the trunk before symptom development have prevented a critical study of
the toxin in vine plants. Determining the gene responsible for eutypine
resistance would therefore be an important critical tool in determining
the role of eutypine toxin in symptom development in the disease; and
it has the potential to confer resistance to transgenic
grapevines.Recently, Colrat et al. (1998) found detoxification to occur in
grapevine cells through the enzymatic reduction of eutypine into its
corresponding alcohol, eutypinol
(4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol). We have
determined that this derivative of the toxin is nontoxic for grapevine
tissues. Furthermore, we have established a relationship between the
susceptibility of grapevine to eutypa dieback and the ability of
tissues to inactivate eutypine, suggesting that the detoxification
mechanism plays an important role in defense reactions. Eutypine is
enzymatically detoxified in numerous plant species and, among them, we
found that the tissues of mung bean (Vigna radiata), a
nonhost plant for the pathogen, exhibit an efficient detoxification
activity. As a prerequisite for demonstrating the involvement of
eutypine toxin in eutypa dieback, we report here the purification to
homogeneity and the characterization of an ERE from etiolated mung bean
hypocotyls. 相似文献
2.
The ascomycete Eutypa lata is the causative agent of eutypa dieback in grapevines, a serious economic problem in major wine grape producing areas. In order to develop a predictive, non-destructive assay for early detection of fungal infection, the phenolic metabolite profiles of 11 strains of E. lata grown on four different artificial growth media were analyzed by HPLC and their variability compared with growth on Cabernet Sauvignon grapevine wood and wood extracts. Six compounds were generally produced in significant amounts, namely eutypinol, eulatachromene, and eutypine and its benzofuran cyclization product, together with siccayne and eulatinol. The two most widely distributed and abundant metabolites were eutypinol and eulatachromene, which were present in 8 of the strains grown on grapewood aqueous extract fortified with sucrose. Metabolite production on grapevine extract was greatly enhanced relative to the artificial media, indicating that this native substrate provides optimal conditions and a more representative profile of the metabolites produced in the natural disease state. The primary metabolites were tested in a grapeleaf disc bioassay to establish their relative toxicity. Neither eutypinol nor siccayne were phytotoxic; eulatachromene, eulatinol, eutypine, and the benzofuran exhibited necrotic effects in the bioassay. The results indicate that eutypa dieback may be caused by several E. lata metabolites rather than a single compound. 相似文献
3.
S. Colrat C. Deswarte A. Latché A. Klaébé M. Bouzayen J. Fallot J. P. Roustan 《Planta》1999,207(4):544-550
Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata (Pers.: Fr.) Tul., the causal agent of dying arm disease of Vitis vinifera L. (grapevine). Previously, we have shown that eutypine is involved in the development of disease symptoms. In the present
study, the effects of V. vinifera cell-suspension cultures on the biological activity of the toxin were investigated. Eutypine was converted by grapevine tissues
into a single compound, identified by mass spectrometry and nuclear magnetic resonance as 4-hydroxy-3-(3-methyl-3-butene-1-ynyl)
benzyl alcohol, designated eutypinol. This compound was found to be non-toxic for grapevine tissues. Unlike eutypine, eutypinol
failed to affect the oxidation rate or membrane potential of isolated mitochondria. In grapevine cells, reduction of eutypine
into the corresponding alcohol is an NADPH-dependent enzymatic reaction. An enzyme which reduced eutypine was partially purified,
over 1000-fold, using a five-step purification procedure. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, the protein was found to have a molecular mass of 54–56 kDa. The enzyme exhibited an apparent K
m for eutypine of 44 μM, and was active between pH 6.8 and 7.5 with a maximum at pH 7.0. The eutypine reductase activity was
improved by Mn2+ and Mg2+ and inhibited by disulfiram and p-hydroxymercuribenzoate. The possible role of the eutypine-detoxification mechanism in the defense reactions of V. vinifera cells is discussed.
Received: 20 April 1998 / Accepted: 22 September 1998 相似文献
4.
Acetylenic phenols and a chromene isolated from the grapevine fungal pathogen Eutypa lata were examined for mode of toxicity. The compounds included eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl aldehyde), eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), eulatachromene, 2-isoprenyl-5-formyl-benzofuran, siccayne, and eulatinol. A bioassay using the yeast Saccharomyces cerevisiae showed that all compounds were either lethal or inhibited growth. A respiratory assay using 2,3,5-triphenyltetrazolium (TTC) indicated that eutypinol and eulatachromene inhibited mitochondrial respiration in wild-type yeast. Bioassays also showed that 2-isoprenyl-5-formyl-benzofuran and siccayne inhibited mitochondrial respiration in the S. cerevisiae deletion mutant vph2, lacking a vacuolar type H (+) ATPase (V-ATPase) assembly protein. Cell growth of tsa1, a deletion mutant of S. cerevisiae lacking a thioredoxin peroxidase (cTPx I), was greatly reduced when grown on media containing eutypinol or eulatachromene and exposed to hydrogen peroxide (H2O2) as an oxidative stress. This reduction in growth establishes the toxic mode of action of these compounds through inhibition of mitochondrial respiration. 相似文献
5.
E. Chevreau F. Dupuis J. P. Taglioni S. Sourice R. Cournol C. Deswartes A. Bersegeay J. Descombin M. Siegwart K. Loridon 《Plant Cell, Tissue and Organ Culture》2011,106(1):161-168
The development of alternative selection systems without antibiotic resistance genes is a key issue to produce safer and more
acceptable transgenic plants. Eutypine is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevine, which is detoxified in mung bean (Vigna radiata) by the gene Vr-ERE. Many phytotoxic compounds containing an aldehyde group can act as substrates for the Vr-ERE enzyme. The aim of the present
work was to evaluate the effects of the overexpression of Vr-ERE in transgenic apple plants, as a first step towards the development of an alternative selection system. Viable transgenic
apple clones expressing Vr-ERE were produced from the cultivar Greensleeves under kanamycin selection. Although the Vr-ERE transgene was normally expressed at the RNA and protein levels, the increase in aldehyde reductase activity tested on a range
of potential substrates was very low in these clones. None of them revealed a significant increase in tolerance to toxic aldehydes
compared to their non-transgenic control. This work with transgenic apple plants overexpressing the detoxifying gene Vr-ERE illustrates some of the difficulties in developing an alternative selection pressure. 相似文献
6.
Control of anthocyanin biosynthesis pathway gene expression by eutypine,a toxin from Eutypa lata,in grape cell tissue cultures 总被引:1,自引:0,他引:1
Afifi M El-Kereamy A Legrand V Chervin C Monje MC Nepveu F Roustan JP 《Journal of plant physiology》2003,160(8):971-975
Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of Eutypa dieback in grapevine. The effect of the toxin on anthocyanin synthesis has been investigated in Vitis vinifera cv. Gamay cell cultures. At concentrations higher than 200 micromol/L, eutypine reduced anthocyanin accumulation in cells. The reduction in anthocyanin accumulation was proportional to the eutypine concentrations and HPLC analysis showed that eutypine affected the levels of all anthocyanins. The effect of eutypine application on the expression of five genes of the anthocyanin biosynthesis pathway, including chalcone synthase (CHS), flavonone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT) was determined. Expression of CHS, F3H, DFR and LDOXwas not affected by the addition of eutypine to grapevine cell cultures. In contrast, expression of the UFGT gene was dramatically inhibited by the toxin. These results suggest that in grapevine cell cultures, eutypine strongly affects anthocyanin accumulation by inhibiting UFGT gene expression. The mechanism of action of eutypine is discussed. 相似文献
7.
Generation of reactive oxygen species (ROS) in plants is an inevitable consequence of adverse environmental cues and the ability to detoxify deleterious by-products of ROS-mediated oxidation reactions reflect an important defence strategy to combat abiotic stress. Here, we have cloned the eutypine reducing aldehyde reductase gene (VrALR) from Vigna radiata (L.) Wilczek roots. We have expressed and purified the VrALR protein and analyzed its enzyme kinetic parameters and catalytic efficiency with three different substrates to confirm its identity. The functional characterization of this enzyme was unravelled through heterologous expression of the gene in Escherichia coli BL21 and an oxidative stress-sensitive Saccharomyces cerevisiae mutant strain, W3O3-1-A. Finally, the endogenous VrALR enzyme activity and the mRNA expression patterns of the VrALR gene in the roots of V. radiata in response to progressive drought stress in vivo was studied to correlate the ROS-detoxifying role of this important enzyme under the influence of progressive drought stress. Our results, for the first time, demonstrate that eutypine reducing VrALR provides varying degree of stress tolerance in bacteria, yeast systems and also plays a promising protective role against oxidative stress in V. radiata roots during gradual water deprivation. The present study provides an unequivocal evidence to understand the crucial role of aldehyde reductase ROS-detoxifying system which is highly essential for developing stress tolerance in economically important crop plants. 相似文献
8.
M. Ramírez-Suero M. Bénard-Gellon J. Chong H. Laloue E. Stempien E. Abou-Mansour F. Fontaine P. Larignon F. Mazet-Kieffer S. Farine C. Bertsch 《Protoplasma》2014,251(6):1417-1426
Three major grapevine trunk diseases, esca, botryosphaeria dieback and eutypa dieback, pose important economic problems for vineyards worldwide, and currently, no efficient treatment is available to control these diseases. The different fungi associated with grapevine trunk diseases can be isolated in the necrotic wood, but not in the symptomatic leaves. Other factors seem to be responsible for the foliar symptoms and may represent the link between wood and foliar symptoms. One hypothesis is that the extracellular compounds produced by the fungi associated with grapevine trunk diseases are responsible for pathogenicity. In the present work, we used Vitis vinifera cv. Chardonnay cells to test the aggressiveness of total extracellular compounds produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with botryosphaeria dieback. Additionally, the toxicity of purified mellein, a characteristic toxin present in the extracellular compounds of Botryosphaeriaceae, was assessed. Our results show that the total extracellular compounds produced by N. parvum induce more necrosis on Chardonnay calli and induce a different defence gene expression pattern than those of D. seriata. Mellein was produced by both fungi in amounts proportional to its aggressiveness. However, when purified mellein was added to the culture medium of calli, only a delayed necrosis and a lower-level expression of defence genes were observed. Extracellular compounds seem to be involved in the pathogenicity of the fungi associated with botryosphaeria dieback. However, the doses of mellein used in this study are 100 times higher than those found in the liquid fungal cultures: therefore, the possible function of this toxin is discussed. 相似文献
9.
Dr. Sabine von Tiedemann Prof. Dr. Günter Brendel Prof. Dr. Hartmut Fehrmann 《Journal of Phytopathology》1988,122(2):147-165
Investigations on endophytic fungi of grapevine with special emphasis on the vascular system of rootstocks In three vegetation periods endophytic fungi from rootstocks of grafted grapevines were isolated, and their influence on the host-plant was determined. Before grafting (pre-nursery) about 40 to 50% of grapevines were colonized by endophytic fungi; in grafted material after growing in a nursery this rate increased up to 50 to 90% (post-nursery). Most of these fungi are Deuteromycetes, especially Moniliales and Coelomycetes. Only a few Ascomycetes were found. The spectrumof fungi differs, mainly depending on the season (pre- and post-nursery), but less on the variety and year of sampling. Contradictory results from investigations of the relationship between the isolated fungi and the vitality of grafted grapevines were found, however the endophytes clearly showed no pathogenic character. On the contrary in some cases a higher vitality of the grapevines correlated with presence of endophytes. 相似文献
10.
《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1997,320(2):149-158
Eutypa dieback, caused by the ascomycet fungus Eutypa lata, is currently the most serious disease of the grapevine. This disease now affects a great number of vineyards throughout the world, its economic impact is very important, and there is no remedy available for the destruction of the parasitic fungus. In this article, we describe the dieback symptoms, the management practices, the economic impact and the present knowledge on the interactions between E. lata and grapevine. We also present findings concerning the role of a toxic compound, hydroxy-4(methyl-3 butene-3 ynyl-1)-3 benzaldehyde, name eutypine, synthesized by the parasitic fungus and which was shown to be involved in the expression of the disease symptoms. The recent progress made in understanding eutypine's mechanism of action has opened new prospects regarding development of efficient tools for stopping this disease. 相似文献
11.
In this study we have examined the roles of alcohol dehydrogenase, aldehyde oxidase, and aldehyde dehydrogenase in the adaptation of Drosophila melanogaster to alcohol environments. Fifteen strains were characterized for genetic variation at the above loci by protein electrophoresis. Levels of in vitro enzyme activity were also determined. The strains examined showed considerable variation in enzyme activity for all three gene-enzyme systems. Each enzyme was also characterized for coenzyme requirements, effect of inhibitors, subcellular location, and tissue specific expression. A subset of the strains was chosen to assess the physiological role of each gene-enzyme system in alcohol and aldehyde metabolism. These strains were characterized for both the ability to utilize alcohols and aldehydes as carbon sources as well as the capacity to detoxify such substrates. The results of the above analyses demonstrate the importance of both alcohol dehydrogenase and aldehyde dehydrogenase in the in vivo metabolism of alcohols and aldehydes. 相似文献
12.
Cultures of F. moniliforme var. subglutinans, F. moniliforme, F. lateritium, F. equiseti, F. semitectum and F. solani from pine and F. moniliforme and F. graminearum from southern U.S. corn were grown on rice and corn, extracted, and checked for toxicity in mice, chicken embryos, and pine seedlings, and for mutagenicity by the Ames test. While extracts from both fungal groups contained toxins, none of the extracts induced dieback in pine seedlings. Almost all of the cultures isolated from corn in contrast to those from pine, were mutagenic. Thin-layer chromatography did not detect T-2 toxin, moniliformin, or vomitoxin, indicating that these toxins do not elicit dieback symptoms in pine. 相似文献
13.
Dieback of Passion Fruit in Surinam 总被引:1,自引:0,他引:1
In Surinam, the commercial cultivation of the yellow passion fruit (Passiflora edulis f. flavicarpa) is difficult due to the occurrence of dieback. Symptoms referred to as dieback include a decrease in elongation of the shoot end internodes after a period of normal growth leading to wilting and death of the shoots. Fruits from plants showing dieback symptoms are much smaller than those from healthy plants. From shoots with dieback symptoms, three fungi were isolated including Colletotrichum gloeosporioides. However, inoculation experiments with these fungi on shoots of vigorously growing plants were negative, even after wound inoculation. It appeared that plants with dieback symptoms had a poorly developed root system, From these roots Fusarium solani was isolated, which appeared to be highly pathogenic to roots of the yellow passion fruit. After inoculation of the roots of 3-month-old plants, roots became infected and the aerial plant parts showed typical dieback symptoms. Plants with their root system reduced either by inoculating with F. solani or by clipping, and subsequently inoculated with C. gloeosporioides on the aerial parts 2 weeks later, showed dieback symptoms and infection by C. gloeosporioides in shoots with these symptoms. Thus, a badly functioning root system, for example caused by infection of F. solani leads to dieback and predisposes plants to infection by C. gloeosporioides. The latter fungus itself is not a primary pathogen of shoots of the yellow passion fruit in Surinam. 相似文献
14.
Mulligan CJ Robin RW Osier MV Sambuughin N Goldfarb LG Kittles RA Hesselbrock D Goldman D Long JC 《Human genetics》2003,113(4):325-336
Enzymes encoded by two gene families, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), mediate alcohol metabolism in humans. Allelic variants have been identified that alter metabolic rates and influence risk for alcoholism. Specifically, ADH1B*47His
(previously ADH2-2) and ALDH2-2 have been shown to confer protection against alcoholism, presumably through accumulation of acetaldehyde in the blood and a resultant 'flushing response' to alcohol consumption. In the current study, variants at ADH1B
(previously ADH2), ADH1C (previously ADH3), and
ALDH2 were assayed in DNA extracts from participants belonging to a Southwest American Indian tribe (n=490) with a high prevalence of alcoholism. Each subject underwent a clinical interview for diagnosis of alcohol dependence, as well as evaluation of intermediate phenotypes such as binge drinking and flushing response to alcohol consumption. Detailed haplotypes were constructed and tested against alcohol dependence and related intermediate phenotypes using both association and linkage analysis. ADH and ALDH variants were also assayed in three Asian and one African population (no clinical data) in order to provide an evolutionary context for the haplotype data. Both linkage and association analysis identified several ADH1C alleles and a neighboring microsatellite marker that affected risk of alcohol dependence and were also related to binge drinking. These data strengthen the support for
ADH as a candidate locus for alcohol dependence and suggest further productive study. 相似文献
15.
Peng X Taki H Komukai S Sekine M Kanoh K Kasai H Choi SK Omata S Tanikawa S Harayama S Misawa N 《Applied microbiology and biotechnology》2006,71(6):824-832
Four genes were isolated and characterized for alcohol dehydrogenases (ADHs) catalyzing the oxidation of aromatic alcohols such as benzyl alcohol to their corresponding aldehydes, one from o-xylene-degrading Rhodococcus
opacus TKN14 and the other three from n-alkane-degrading Rhodococcus
erythropolis PR4. Various aromatic alcohols were bioconverted to their corresponding carboxylic acids using Escherichia
coli cells expressing each of the four ADH genes together with an aromatic aldehyde dehydrogenase gene (phnN) from Sphingomonas sp. strain 14DN61. The ADH gene (designated adhA) from strain TKN14 had the ability to biotransform a wide variety of aromatic alcohols, i.e., 2-hydroxymethyl-6-methylnaphthalene, 2-hydroxymethylnaphthalene, xylene-α,α’-diol, 3-chlorobenzyl alcohol, and vanillyl alcohol, in addition to benzyl alcohol with or without a hydroxyl, methyl, or methoxy substitution. In contrast, the three ADH genes of strain PR4 (designated adhA, adhB, and adhC) exhibited lower ability to degrade these alcohols: these genes stimulated the conversion of the alcohol substrates by only threefold or less of the control value. One exception was the conversion of 3-methoxybenzyl alcohol, which was stimulated sevenfold by adhB. A phylogenetic analysis of the amino acid sequences of these four enzymes indicated that they differed from other Zn-dependent ADHs.The first two authors contributed equally to this work 相似文献
16.
Tadahiko Kajiwara Takahiro Harada Akikazu Hatanaka 《Bioscience, biotechnology, and biochemistry》2013,77(1):243-247
Z-3-Hexenal, a precursor in the biosynthesis of leaf alcohol and leaf aldehyde, was first isolated from fresh tea leaves, Thea sinensis and its structure was confirmed by unequivocal synthetic evidence. 相似文献
17.
M. M. Harmsen C. B. van Solt H. P. D. Fijten 《Applied microbiology and biotechnology》2009,84(6):1087-1094
Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments.
However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity
three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize
foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins
were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated
VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown
that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity. 相似文献
18.
Pseudomonas putida CSV86 utilizes benzyl alcohol via catechol and methylnaphthalenes through detoxification pathway via hydroxymethylnaphthalenes
and naphthaldehydes. Based on metabolic studies, benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH)
were hypothesized to be involved in the detoxification pathway. BADH and BZDH were purified to apparent homogeneity and were
(1) homodimers with subunit molecular mass of 38 and 57 kDa, respectively, (2) NAD+ dependent, (3) broad substrate specific accepting mono- and di-aromatic alcohols and aldehydes but not aliphatic compounds,
and (4) BADH contained iron and magnesium, while BZDH contained magnesium. BADH in the forward reaction converted alcohol
to aldehyde and required NAD+, while in the reverse reaction it reduced aldehyde to alcohol in NADH-dependent manner. BZDH showed low K
m value for benzaldehyde as compared to BADH reverse reaction. Chemical cross-linking studies revealed that BADH and BZDH do
not form multi-enzyme complex. Thus, the conversion of aromatic alcohol to acid is due to low K
m and high catalytic efficiency of BZDH. Phylogenetic analysis revealed that BADH is a novel enzyme and diverged during the
evolution to gain the ability to utilize mono- and di-aromatic compounds. The wide substrate specificity of these enzymes
enables strain to detoxify methylnaphthalenes to naphthoic acids efficiently. 相似文献
19.
Hiraku Sasaki Hiroki Ishikawa Toru Sato Satoshi Sekiguchi Hiromi Amao Eiichi Kawamoto Tetsuya Matsumoto Kazuhiko Shirama 《BMC microbiology》2011,11(1):55
Background
Pasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system. 相似文献20.
Evidence is presented for the existence in Pseudomonas putida of two NAD-linked dehydrogenases that function sequentially to oxidize benzyl alcohol. Induction of muconate lactonizing enzyme, a 3-oxoadipate pathway enzyme, indicated that P. putida oxidized benzyl alcohol to benzoate. Polyacrylamide gel electrophoresis with activity staining and enzymatic assays for an NAD-dependent dehydrogenase both showed that cells contained a single, constitutive alcohol dehydrogenase capable of oxidizing benzyl alcohol. This enzyme was shown to have the same specificity in extracts of glucose-grown as in benzy alcoholgrown cells. An NAD-aldehyde dehydrogenase oxidized benzaldehyde but was most active with normal alkyl aldehydes. This aldehyde dehydrogenase was shown to be induced, by enzymatic assays and by activity staining of polyacrylamide gel electropherograms, not only in cells grown on benzyl alcohol, but also in cells grown on ethanol. These experiments suggested that the aldehyde dehydrogenase was induced by the alcohol being oxidized rather than the substrate aldehyde.In sum, the evidence from enzyme assays and polyacrylamide gel electrophoresis of extracts indicates that Pseudomonas putida catabolizes benzyl alcohol slowly when it is the sole carbon and energy source, by the action of a constitutive, nonspecific, alcohol dehydrogenase and an alcohol-induced, nonspecific aldehyde dehydrogenase to yield benzoate, which is further metabolized via the 3-oxoadipate (beta-ketoadipate) pathway.In memory of R. Y. Stanier 相似文献