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1.
There is no effective chemotherapy against diseases caused by Phytomonas sp., a plant trypanosomatid responsible for economic losses in major crops. We tested three triazolo-pyrimidine complexes [two with Pt(II), and another with Ru(III)] against promastigotes of Phytomonas sp. isolated from Euphorbia characias. The incorporation of radiolabelled precursors, ultrastructural alterations and changes in the pattern of metabolite excretion were examined. Different degrees of toxicity were found for each complex: the platinum compound showed an inhibition effect on nucleic acid synthesis, provoking alterations on the levels of mitochondria, nucleus and glycosomes. These results, together with others reported previously in our laboratory about the activity of pyrimidine derivatives, reflect the potential of these compounds as agents in the treatment of Phytomonas sp.  相似文献   

2.
 The use of genetically engineered crop plants has raised concerns about the transfer of their engineered DNA to indigenous microbes in soil. We have evaluated possible horizontal gene transfer from transgenic plants by natural transformation to the soil bacterium Acinetobacter calcoaceticus BD413. The transformation frequencies with DNA from two sources of transgenic plant DNA and different forms of plasmid DNA with an inserted kanamycin resistance gene, nptII, were measured. Clear effects of homology were seen on transformation frequencies, and no transformants were ever detected after using transgenic plant DNA. This implied a transformation frequency of less than 10-13 (transformants per recipient) under optimised conditions, which is expected to drop even further to a minimum of 10-16 due to soil conditions and a lowered concentration of DNA available to cells. Previous studies have shown that chromosomal DNA released to soil is only available to A. calcoaceticus for limited period of time and that A. calcoaceticus does not maintain detectable competence in soil. Taken together, these results suggest that A. calcoaceticus does not take up non-homologous plant DNA at appreciable frequencies under natural conditions. Received: 1 November 1996 / Accepted: 18 April 1997  相似文献   

3.
To analyse the frequency of natural gene transfer from genetically modified maize to phytopathogenic bacterium Erwinia stewartii 1082, a marker rescue system based on the restoration of ampicillin resistance gene was used in in vitro and in planta transformation experiments. A set of three vectors containing defined deletions of the blaTEM116 ampicillin resistance gene in pBR322 was constructed. Recombinant strains of Erw. stewartii 1082 harboring these mutant plasmids were used for infection of transgenic maize plants. Restoration of ampicillin resistance was observed only in transformed electro-competent Erw. stewartii 1082 cells. Frequency of the resistance restoration was found to be dependent on the size of the transforming DNA. In addition, highly active non-specific endodeoxyribonuclease was detected in cell-free lysates of Erw. stewartii 1082, rapidly degrading linear DNA fragments. No ampicillin resistant Erw. stewartii 1082 transformants were observed during in planta experiments indicating that this pathogenic bacterium is not naturally transformable under the conditions tested in this study.  相似文献   

4.
Stereoselective reduction towards pharmaceutically potent products with multi‐chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH‐dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with this enzyme indicated outstanding potential for the reduction of various alpha‐hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl2. Detailed analysis of the pH‐dependent activity and stability yielded a broad pH‐optimum (pH 6–9.5) for the reduction reaction and a sharp optimum of pH 10–11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5–8 and 8–15°C; nevertheless, biotransformations can also be carried out at 25°C (half‐life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio‐ and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2‐hydroxypropiophenone in more detail. Biotechnol. Bioeng. 2013; 110: 1838–1848. © 2013 Wiley Periodicals, Inc.  相似文献   

5.
Toluene and related aromatic compounds are anaerobically degraded by the denitrifying bacterium Thauera sp. strain K172 via oxidation to benzoyl-CoA. The postulated initial step is methylhydroxylation of toluene to benzyl alcohol, which is either a free or enzyme-bound intermediate. Cells grown with toluene or benzyl alcohol contained benzyl alcohol dehydrogenase, which is possibly the second enzyme in the proposed pathway. The enzyme was purified from benzyl-alcohol-grown cells and characterized. It has many properties in common with benzyl alcohol dehydrogenase from Acinetobacter and Pseudomonas species. The enzyme was active as a homotetramer of 160kDa, with subunits of 40kDa. It was NAD+-specific, had an alkaline pH optimum, and was inhibited by thiol-blocking agents. No evidence for a bound cofactor was obtained. Various benzyl alcohol analogues served as substrates, whereas non-aromatic alcohols were not oxidized. The N-terminal amino acid sequence indicates that the enzyme belongs to the class of long-chain Zn2+-dependent alcohol dehydrogenases, although it appears not to contain a metal ion that can be removed by complexing agents.Dedicated to Prof. Achim Trebst  相似文献   

6.
Investigation of the stereochemistry of the hydride transfer in reactions catalyzed by the recently isolated NAD+-linked alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123 was accomplished by using 1H NMR spectroscopy of the deuterated coenzyme. It was found that this new psychrophilic enzyme is a type A dehydrogenase. Moraxella sp. ADH reduces stereospecifically 2-butanone to produce (S)-2-butanol.  相似文献   

7.
Summary Naturally occurring horizontal gene transfers between nonviral organisms are difficult to prove. Only with the availability of sequence data from a wide variety of organisms can a convincing case be made. In the case of putative gene transfers between prokaryotes and eukaryotes, the minimum requirements for inferring such an event include (1) sequences of the transferred gene or its product from several appropriately divergent eukaryotes and several prokaryotes, and (2) a similar set of sequences from the same (or closely related organisms) for another gene or genes. Given these criteria, we believe that a strong case can be made forEscherichia coli having acquired a second glyceraldehyde-3-phosphate dehydrogenase gene from some eukaryotic host. Ancillary observations on the general rate of change and the time of the prokaryote-eukaryote divergence support the notion.  相似文献   

8.
M Peretz  Y Burstein 《Biochemistry》1989,28(16):6549-6555
The complete amino acid sequence of alcohol dehydrogenase of Thermoanaerobium brockii (TBAD) is presented. The S-carboxymethylated protein was cleaved at methionine residues (with cyanogen bromide) to provide a set of 10 nonoverlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by proteolytic cleavage at lysine residues (with Achromobacter protease I). The protein subunit contained 352 amino acid residues corresponding to a molecular weight of 37,652. The sequence showed about 35% identity with that of the prokaryotic Alcaligenes eutrophus alcohol dehydrogenase and about 25% identity with any one of the eukaryotic alcohol/polyol dehydrogenases known today. Of these, only 18 residues (5%) are strictly conserved: 11 Gly, 2 Asp, and 1 each of Cys, His, Glu, Pro, and Val.  相似文献   

9.
The nucleotide sequence of the alcohol dehydrogenase gene Adh71k has been determined. The Adh71k allele encodes the thermostable and multifunctional ADH-71k allozyme of Drosophila melanogaster. Comparison with the sequences of AdhS, AdhF, and AdhFChD reveals differences in the coding and noncoding regions of the gene. Conceptual translation of the Adh71k sequence indicates that ADH-71k shares with ADH-F and ADH-FCHD an amino acid replacement at residue 192 and with ADH-FCHD an additional replacement of serine for proline at residue 214. Three unique differences were found in the nontranslated regions. It is proposed that a nucleotide deletion in the adult intron is related to the difference in expression level of the Adh71k allele, relative to the other alleles. An insertion of five nucleotides, additional to a single base deletion at that site, was detected in one of the larval enhancer regions in the 5' flanking region of the Adh71k allele, creating a palindromic structure in that area.  相似文献   

10.
Capsular polysaccharides are important virulence factors both in Gram-positive and Gram-negative bacteria. A similar cluster organization of the genes involved in the synthesis of bacterial exopolysaccharides has been postulated in both cases, suggesting that these clusters evolved by module assembly. Horizontal gene transfer has been postulated to explain the polymorphism found in these cellular polymers. The cap1K and cap3A genes coding for the pneumococcal type 1 and type 3 UDP-glucose dehydrogenases, respectively, have been compared with other UDP-sugar dehydrogenases. We have observed that the evolutionary distance between Cap1K and Cap3A is approximately equal to that found between Cap1K (or Cap3A) and other UDP-GlcDH of families evolutionarily distant like KfiD, the dehydrogenase from Escherichia coli K5. On the basis of comparisons of G + C content, patterns of synonymous and nonsynonymous substitutions, dinucleotide frequencies, and codon usage bias, we conclude that the kfiD gene has been introduced into E. coli from an exogenous source, probably from a streptococcal species. Received: 26 May 1997 / Accepted: 30 July 1997  相似文献   

11.
Summary Membrane-bound alcohol dehydrogenase (ADH) was purified from the membrane fraction of an industrial-vinegar-producing strain, A. polyoxogenes sp. nov. NBI1028 by solubilization using Triton X-100 and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis (PAGE). Upon sodium dodecyl sulphate-PAGE, the enzyme showed the presence of two subunits with a molecular mass of 72 000 daltons and 44 000 daltons, respectively. The small subunit was identified as cytochrome c. In addition, absorption and fluorescence spectra showed the the presence of pyrroloquinoline quinone in the purified ADH. The ADH preferentially oxidized aliphatic alcohols with a straight carbon chain except for methanol. Formaldehyde and acetaldehyde were also oxidizable substrates. The apparent K m for ethanol was 1.2 mM. The optimum pH and temperature were 5.0–6.0 and 40°C, respectively. p-Chloromercuribenzoic acid and heavy metals such as CuSO4 were inhibitory to the enzyme activity. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya  相似文献   

12.
An NAD-dependent secondary alcohol dehydrogenase (ADH) produced by Rhodococcus sp. GK1 was purified about fivefold with a yield of 82% by hydrophobic interaction chromatography. This enzyme reduced monoketones, diketones and α-dicarbonyl compounds ; it oxidized secondary alcohols but not primary alcohols. Optimum pH was 7·0 or 8·5 for reduction or oxidation of substrates, respectively, and optimal temperature for activity was 55 °C. The apparent molecular mass of ADH was about 60 kDa by gel filtration chromatography.  相似文献   

13.
1. No primary hydrogen acceptor other than phenazine methosulphate has been found for the alcohol dehydrogenase from Pseudomonas sp. M27. 2. None of a wide range of vitamins or cofactors has any effect on the activity of the enzyme. 3. The enzyme is far less sensitive to metal-chelating agents and thiol reagents than are other alcohol dehydrogenases. 4. Methanol is oxidized at least as fast as other alcohols by this enzyme and its well-defined substrate specificity is different from that of other alcohol dehydrogenases. Only primary alcohols are oxidized; the general formula for an oxidizable substrate is R.CH(2).OH, where R may be H or [Formula: see text] 5. Whole organisms oxidize only those alcohols that are oxidized by the isolated enzyme.  相似文献   

14.
Quantitative trait locus (QTL) analysis was used to study the evolution of genes controlling the timing of flowering in four Brassica genomes that are all extensively replicated. Comparative mapping showed that a chromosomal region from the top of Arabidopsis thaliana chromosome 5 corresponded to three homoeologous copies in each of the diploid species Brassica nigra, B. oleracea, and B. rapa and six copies in the amphidiploid B. juncea. QTLs were detected in two of the three replicated segments in each diploid genome and in three of the six replicated segments in B. juncea. These results indicate that, for the studied trait, multiple QTLs resulting from genome duplication is the rule rather than the exception. Brassica homologues to two candidate genes (CO and FLC) identified from the corresponding A. thaliana region were mapped. CO homologues mapped close to the QTL peaks in eight of nine QTLs, while FLC homologues mapped farther away in those cases where the mapping resolution allowed a comparison. Thus, our data are consistent with the hypothesis that all the major QTLs we detected in the different species of Brassica could be the result of duplicated copies of the same ancestral gene, possibly the ancestor of CO.  相似文献   

15.
16.
Centrifugation through a cesium chloride density gradient and agarose gel electrophoresis of the DNA from the purple non-sulfur photosynthetic bacterium Ectothiorhodospira sp. resolved a single extrachromosomal element, plasmid pDG1. Its size was estimated to be 13.2 kilobases by restriction endonuclease mapping. Plasmid pDG1 and two restriction fragments thereof were cloned in Escherichia coli C600 with plasmid pBR327 as a vector to form mixed plasmids pDGBR1, pDGBR2, and pDGBR3. The resistance to streptomycin and mercury found in Ectothiorhodospira sp. was transferred to E. coli C600 after transformation with pDGBR1 but not with pDGBR2 and pDGBR3. The replication origin of pDG1 was estimated to be within a 2-kilobase restriction fragment of pDG1 by monitoring its replication in E. coli HB101, using a kanamycin resistance reporter gene. High stringency molecular hybridization with 32P-labeled pDG1 identified specific fragments of genomic DNA, suggesting the integration of some plasmid sequences. In accordance with the hypothesis that this integration is due to a transposon, we tested the transfer of streptomycin resistance from pDG1 into plasmid pVK 100 used as a target. For this test, we regrouped in the same cells of E. coli HB101, pDGBR1 and mobilizable plasmid pVK100 (tetr, kmr). We used the conjugation capacity of the pVK100/pRK2013 system to rescue the target plasmid pVK100 into nalidixic acid-resistant E. coli DH1. The transfer frequency of streptomycin resistance into pVK100 was 10−5, compatible with a transposition event. In line with the existence of a transposon on pDG1, heteroduplex mapping indicated the presence of inverted repeats approximately 7.5 kb from one another.  相似文献   

17.
18.
19.
To find microorganisms that could reduce phenyl trifluoromethyl ketone (PTK) to (S)-1-phenyltrifluoroethanol [(S)-PTE], styrene-assimilating bacteria (ca. 900 strains) isolated from soil samples were screened. We found that Leifsonia sp. strain S749 was the most suitable strain for the conversion of PTK to (S)-PTE in the presence of 2-propanol as a hydrogen donor. The enzyme corresponding to the reaction was purified homogeneity, characterized and designated Leifsonia alcohol dehydrogenase (LSADH). The purified enzyme had a molecular weight of 110,000 and was composed of four identical subunits (molecular weight, 26,000). LSADH required NADH as a cofactor, showed little activity with NADPH, and reduced a wide variety of aldehydes and ketones. LSADH catalyzed the enantioselective reduction of some ketones with high enantiomeric excesses (e.e.): PTK to (S)-PTE (>99% e.e.), acetophenone to (R)-1-phenylethanol (99% e.e.), and 2-heptanone to (R)-2-heptanol (>99% e.e.) in the presence of 2-propanol without an additional NADH regeneration system. Therefore, it would be a useful biocatalyst.  相似文献   

20.
The gene of NAD+-dependent formate dehydrogenase (FDH) from Mycobacterium vaccae N10 was cloned into Escherichia coli by hybridization with digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain reaction with degenerate primers. The primers were designed on the basis of the most conserved parts of known sequences of FDH from different organisms. An open-reading frame of 1200 bp exhibited extremely high sequence similarity to the FDH gene of Pseudomonas sp. 101. The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was identical to that of Pseudomonas sp. 101 (PsFDH) except for two amino acid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lysine in PsFDH). The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH. To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate-61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by site-directed mutagenesis. All the mutant enzymes showed higher thermostability than the wild-type McFDH. The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.  相似文献   

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