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1.
2.
Nopaline (N-[4-[(aminoiminomethyl)amino-]-1S-carboxybutyl]-2R-aminopentanedioic acid and isonopaline (N-[4-[(aminoiminomethyl)amino-1S-carboxybutyl]- 2S-aminopentanedioic acid) have been synthesized and separated by crystallization. In addition, a derivative of each of these compounds that forms spontaneously from the parent compounds under the usual crystallization conditions was isolated and characterized. The chemical properties, elemental analysis, 1H-NMR spectrum, and electrophoretic behavior of the derivative from nopaline are consistent with N-[4-[ (aminoiminomethyl)amino]-1S-carboxybutyl]-2-pyrrolidone-5R-carboxylic acid, also called pyronopaline. The presence of pyronopaline in crown gall tumor tissue and the catabolism of it by the bacterium A. tumefaciens establish it as a new opine.  相似文献   

3.
Succinamopine: a new crown gall opine   总被引:10,自引:5,他引:5       下载免费PDF全文
Agrobacterium tumefaciens strains can incite plant tumors consisting of transformed cells that synthesize novel metabolites called opines. The pattern of opine synthesis is dictated by plasmid-borne genes in the pathogen; additional plasmid genes confer on the pathogen the ability to catabolize the same pattern of opines synthesized. One group of A. tumefaciens strains, AT181, EU6, and T10/73, contains closely related tumor-inducing (Ti) plasmids that encode the ability to degrade the opine nopaline; but tumors incited by these strains do not synthesize nopaline. We demonstrated by Southern blot hybridization that AT181(pTi) has no DNA homologous to the nopaline synthase gene of pTi T37, a nopaline Ti plasmid that appears to be most closely related to this group based on fingerprint analysis. Tumors incited by these seemingly anomalous strains contain a new opine that we designate succinamopine. Its structure is analogous to that of nopaline, with asparagine replacing arginine. Evidence for the structure of succinamopine, as well as those of two related metabolites, succinamopine lactam and succinopine lactam, will be published elsewhere. Ability to catabolize succinamopine, succinamopine lactam, and succinopine lactam is encoded by pTi AT181, pTi EU6, and pTi T10/73, but not by any of 15 other Ti and root-inducing plasmids tested. Three avirulent strains tested did not catabolize succinamopine, succinamopine lactam, or succinopine lactam. We propose that pTi AT181, pTi EU6, and pTi T10/73 be designated the succinamopine Ti plasmids.  相似文献   

4.
K. Kamo 《Plant cell reports》1997,16(6):389-392
Five tumorigenic strains ofAgrobacterium tumefaciens were used to inoculate corms, cormels, plants grown in vitro, and seed-derived seedlings of several cultivars ofGladiolus. Tumors formed on 12% of the plant tissues inoculated, and 1% of these tumors synthesized either octopine or nopaline.A. tumefaciens-mediated-glu-curonidase (GUS) expression showed 0.5% and 3.5% GUS expression for plants grown in vitro and regenerable callus, respectively. GUS expression ranged from 40% to 61% whenA. tumefaciens was incubated with leaves from seedlings grown in the dark, whereas leaves from seedlings grown under a 16-h light photoperiod showed no GUS, indicating the significant effect of etiolation on transient GUS expression mediated byA. tumefaciens.  相似文献   

5.
6.
Alfalfa tumour incited by Agrobacterium tumefaciens strain A281, carrying the tumour inducing plasmid pTi Bo542, synthesizes agropine and related mannityl opines. In addition it contains a small amount of leucinopine and large quantities of a new opine here identified as N-[(1S)-1-carboxy 2-carbamoylethyl]-(S)-glutamic acid. This new opine, L,L-succinamopine, is the Lglu epimer of the succinamopine previously isolated from tumours incited by pTi AT181 and related strains. The latter opine should now be designated D,L-succinamopine. This is the first example of the natural occurrence of epimeric opine structures.  相似文献   

7.
Flavin-containing opine dehydrogenase from Bradyrhizobium japonicum forms a heterooligomeric α4β4γ4 enzyme complex. An electron paramagnetic resonance spectroscopy analysis using wild-type and site-directed mutants revealed that [4Fe-4S] and [2Fe-2S] clusters bind to two different types of [Fe-S] binding sites in the γ- and α-subunits, respectively. The latter was found to be important for structural folding and enzyme catalysis.  相似文献   

8.
Mannityl opine accumulation and exudation by transgenic tobacco   总被引:1,自引:0,他引:1       下载免费PDF全文
Three genes from the TR region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants.  相似文献   

9.
Summary Plant cells neoplastically transformed by insertion of T-DNA from Ti plasmids of agrobacteria produce and secrete opines. Secretion of two of these opines (octopine and nopaline) is shown to require the expression of a single gene, which we designate ons.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal  相似文献   

10.
Summary In order to elucidate the regulatory parameters which determine multiple opine formation in marine invertebrates, anaerobiosis was induced in 25 species from several phyla by stimulating the animals to vigorous muscular activity or by subjecting them to environmental hypoxia. The quantity of glycolytic end products and the corresponding amino acids were measured. In a second set of experiments the amounts of substrates and products of the opine dehydrogenase reactions in the isolated introvert retractor muscle (IRM) ofSipunculus nudus were determined in both situations.During environmental hypoxia opines accumulated according to the contents of the corresponding amino acids. Mass action ratios (MAR) of the opine dehydrogenase reactions in the isolated IRM were in the range of control values (octopine dehydrogenase 1.9·1011 mol–2·l2, strombine dehydrogenase 2.2·1010 mol–2·l2). During muscular activity those opines accumulated preferentially which corresponded to the highest opine dehydrogenase activities. In the isolated IRM only octopine accumulated during contractile activity; the MAR of the octopine dehydrogenase reaction was near the control value while the MAR of the strombine dehydrogenase reaction deviated by a factor of 9.The results indicate that during environmental hypoxia the opine dehydrogenases present in a tissue catalyze near equilibrium and the relative amount of opines accumulated is dictated by the concentration of the corresponding amino acids. During muscular activity only those opine dehydrogenases catalyze near equilibrium which are present in sufficiently high activities to keep pace with an increased glycolytic flux. Therefore, different opines may accumulate in the same animal during muscular activity and during environmental hypoxia.  相似文献   

11.
Crown gall tumors incited by Agrobacterium tumefaciens strain Bo542 have been reported to synthesize a tumor-specific substance identified as N-(1,3-dicarboxypropyl)-leucine (leucinopine), a compound with two centers of asymmetry. We report here evidence that leucinopine is indeed a crown gall opine, in that it is specifically catabolized by A. tumefaciens strains carrying the tumor-inducing plasmid pTi Bo542, as well as strains carrying closely related plasmids pTi AT1 and pTi AT4. We further report catabolism of leucinopine by the succinamopine-type strains A518, A519 and A532, carrying pTi EU6, pTi AT181 and pTi T10/73, respectively. Strains lacking any virulence plasmid, as well as those carrying octopine or nopaline type Ti plasmids or mannopine type Ri plasmids, did not catabolize leucinopine. On the basis of specificity of catabolism by bacteria carrying pTi Bo542, we conclude that the stereochemistry of natural leucinopine is l-threo, i.e. lglu,lleu. Such stereochemistry is novel in the opines known thus far: octopine, nopaline and succinamopine have d,l-stereochemistry: dala,larg (octopine), dglu,larg (nopaline) and dglu,lasn (succinamopine).  相似文献   

12.
Previous studies have shown that Nicotiana tabacum contains three Agrobacterium‐derived T‐DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2′ gene (TB‐mas2′). This gene is similar to the Agrobacterium rhizogenes A4‐mas2′ gene that encodes the synthesis of the Amadori compound deoxyfructosyl‐glutamine (DFG or santhopine). In this study we show that TB‐mas2′ is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB‐mas2′ expression levels. The TB‐mas2′ promoter sequences of low‐ and high‐expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low‐ and a high‐expression cultivar. pTB‐mas2‐GUS and pA4‐mas2‐GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB‐mas2′ expression contain detectable levels of DFG.  相似文献   

13.
  • 1.1. Opine dehydrogenases (OpDHs) and lactate dehydrogenase (LDH) activities were determined in various marine animals. OpDHs were detected in six marine invertebrate phyla; Porifera, Coelenterata, Annelida, Mollusca, Arthropoda and Echinodermata in phylogenic sequence.
  • 2.2. Among several OpDHs, tauropine dehydrogenase (TaDH) occurred widely in marine invertebrates, from Porifera to Echinodermata.
  • 3.3. With a few exceptions, total OpDHs activities exceeded that of LDH activity in the marine invertebrates investigated.
  • 4.4. With respect to anaerobic glycolysis, OpDHs are indicated to play an important role in phylogenically lower invertebrates, whereas LDH is more important in higher animals.
  相似文献   

14.
Five virulent Agrobacterium spp. strains that can catabolize the mannityl opines mannopine (MOP), mannopinic acid ( MOA ), and agropinic acid (AGA) were tested for their ability to grow on analogs of these compounds. Analogs containing alternative amino acids replacing glutamic acid or glutamine were generally refused by these bacteria, but mutants were obtained that catabolized the entire family of analogs. In the case of strain C58C1 (pRi 8196), we demonstrated that typical mutants were constitutive for MOP uptake, whereas the wild-type parent was inducible by MOP. Analogs of MOA prepared from a variety of sugars instead of mannose were generally refused, except for a strain carrying pTi B6-806, which grew well on all such analogs. The analogs allowed selection of mutants of all strains. Although most wild-type strains were inducible for AGA uptake, typical mutants selected from strain C58C1 (pRi 8196) were found to be constitutive for uptake of AGA, as was the wild-type strain carrying pTi B6-806. Such constitutive mutants grew on all sugar analogs of MOP, MOA , and AGA tested. The pTi B6-806-containing strain was tested for growth on a more extended series of analogs, including tetrose , triose, diose , and disaccharide analogs, all of which were accepted. Only ketose analogs were refused. Selection of promiscuous regulatory mutants by the two types of opine analogs suggests that the repressor proteins of MOP and AGA permease/ catabolase systems are chiefly responsible for the specificity of the pathways.  相似文献   

15.
Summary The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 15955 was investigated. A partial HindIII digest of pTi15955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli.  相似文献   

16.
We showed that phosphorus-containing metabolites of crown gall tissues were all taken up by appropriate pTi+ agrobacteria. All but one were also taken up by pTi- bacteria. This one compound, produced by nopaline-, but not by octopine-type tumours, was the only phosphorylated organic compound actively secreted by healthy crown gall cells, and it appears to be agrocinopine A. Testing crown gall cell exudates may be a general procedure for the identification of opines by transformed plant cells.  相似文献   

17.
18.
Y Asano  K Yamaguchi    K Kondo 《Journal of bacteriology》1989,171(8):4466-4471
A new NAD+-dependent opine dehydrogenase was purified to homogeneity from Arthrobacter sp. strain 1C isolated from soil by an enrichment culture technique. The enzyme has a molecular weight of about 70,000 and consists of two identical subunits with molecular weights of about 36,000. The enzyme catalyzed a reversible oxidation-reduction reaction of opine-type secondary amine dicarboxylic acids. In the oxidative deamination reaction, the enzyme was active toward unusual opines, such as N-[1-R-(carboxyl)ethyl]-S-methionine and N-[1-R-(carboxyl)ethyl]-S-phenylalanine. In the reductive secondary amine-forming reaction with NADH as a cofactor, the enzyme utilized L-amino acids such as L-methionine, L-isoleucine, L-valine, L-phenylalanine, L-leucine, L-alanine, and L-threonine as amino donors and alpha-keto acids such as pyruvate, oxaloacetate, glyoxylate, and alpha-ketobutyrate as amino acceptors. The product enzymatically synthesized from L-phenylalanine and pyruvate in the presence of NADH was identified as N-[1-R-(carboxyl)ethyl]-S-phenylalanine.  相似文献   

19.
Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.  相似文献   

20.
Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor.  相似文献   

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