Octopine dehydrogenase from Pecten maximus: steady-state mechanism

The steady-state kinetic mechanism of the reaction catalyzed by octopine dehydrogenase [N2-(1-carboxyethyl)-L-arginine:NAD+ oxidoreductase] was investigated at pH 6.9 and 9.2 by studies of substrate inhibition, analogue inhibition, and product inhibition. In the direction of octopine synthesis, the inhibition patterns in the presence of delta- guanidinovalerate and propionate show that NADH binds to the enzyme first followed by L-arginine and pyruvate which bind randomly. In the direction of octopine oxidation, the substrate patterns show that NAD binds to the enzyme before octopine in a rapid equilibrium fashion, and the product inhibition patterns show that the products L-arginine and pyruvate are released in a random fashion. Double, synergistic, substrate inhibition by L-arginine and pyruvate was shown to be due to binding (hypothetically of the imine) to the free enzyme and the enzyme-NAD complex. Furthermore, an alternate minor pathway was demonstrated which includes an enzyme-NADH-octopine complex and an enzyme-octopine complex.     

Octopine Accumulation Early in Crown Gall Development is Progressive

A purification of octopine from crown gall tissue was developed to quantitate conversion of precursor [³H]arginine into [³H]octopine. Plant wound tissue which was sterile or infected with an avirulent strain of Agrobacterium tumefaciens did not accumulate detectable quantities of octopine, consistent with opine synthesis not being induced by wounding or infection. Octopine was only recovered from tissue infected with virulent tumor-inducing strains of A. tumefaciens. In every case tested, the morphological appearance of tumors preceded the accumulation of octopine by at least 1 week, and in some instances 3 weeks. Thus, what was necessary and sufficient for the expression of plant hormones (auxin and cytokinin) required for tumor growth was not sufficient for the accumulation of octopine. The possible nature of the temporal difference in the expression of hormone autotrophy and octopine synthesis is discussed.     

Specificity of Octopine Uptake by Rhizobium and Pseudomonas Strains

The octopine-utilizing strain Agrobacterium tumefaciens B6S3 and three nonagrobacteria which had the capacity to utilize this opine were compared for octopine uptake. The characteristics of uptake by Rhizobium meliloti A3 and strain B6S3 were similar. In both bacteria, uptake activity was inducible by octopine and by the related opine octopinic acid, and competition assays showed that these two opine substrates were accepted by the same uptake system with an equivalent affinity. Cells of Pseudomonas putida 203 accumulated octopine against a concentration gradient, and this activity was induced specifically by octopine. While strain 203 did not utilize octopinic acid, a spontaneous mutant with a combined capacity for octopine and octopinic acid utilization was obtained. Both opines induced octopine uptake by this mutant, but octopinic acid was not a substrate for the induced system. Thus, the Pseudomonas uptake system exhibited a different specificity for octopine than the corresponding Agrobacterium system. The nonfluorescent pseudomonad GU187j, which utilized the three related opines octopine, octopinic acid, and nopaline, was constitutive for octopine uptake. Strain GU187j possessed a system which accepted these three opines, but not arginine or ornithine, with a similar affinity.     

Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase

A single enzyme catalyzes the synthesis of all four N²-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N²-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (K_m) differ, the ratio V/K_m is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Competition of Octopine-Catabolizing Pseudomonas spp. and Octopine-Type Agrobacterium tumefaciens for Octopine in Chemostats

The ability of two octopine-catabolizing Pseudomonas spp. and two virulent octopine-type Agrobacterium tumefaciens to compete for substrates has been examined in chemostats. In dual cultures with octopine or glutamate as the limiting carbon or nitrogen source, Pseudomonas fluorescens B99A and E175D always dominated over A. tumefaciens B6 or ATCC 15955. The growth dynamics of each strain in pure culture indicated that some form of antagonism was occurring in dual culture to permit the predominance of the pseudomonads under certain conditions. Although both pseudomonads fluoresce, pyoverdine was not responsible for the observed inhibition. An unidentified antibiotic secreted by both pseudomonads is believed to be responsible. A. tumefaciens B6 grew synergistically in the presence of P. fluorescens B99A with octopine as the limiting nitrogen source. This behavior of Agrobacterium strain B6 may help overcome its grossly inefficient use of octopine as previously reported. The ability of these two pseudomonads to outcompete the agrobacteria under all conditions tested raises the possibility that under field conditions, infectious agrobacteria may be succeeded by opine-catabolizing pseudomonads around crown gall tumors and in the rhizosphere.     

Octopine Ti-plasmid deletion mutants of agrobacterium tumefaciens with emphasis on the right side of the T-region

One hundred and twelve Agrobacterium tumefaciens mutants with a deleted octopine Ti plasmid were isolated. They originated from four insertion mutants, each of which carried the transposon Tn904 at a different position in the Ti plasmid. The deletion mutants were selected on the basis of loss of the capacity encoded by the Ti plasmid to degrade octopine. They were tested for the expression of other Ti-plasmid coded functions: tumor induction, presence of lysopine dehydrogenase activity in the tumor, and exclusion of phage Ap-1. For 21 mutants affected in at least one of these functions, the map position of the deletions was determined. It was found that deletions at two separated loci give rise to an Occ^? phenotype. Genes for Ap-1 exclusion were mapped on a small region just outside and to the right of the T_L + T_R region. Most of the T_R region, present as T_R-DNA in a limited number of crown gall tissues only, was shown to be unnecessary for tumor formation, since it could be deleted without affecting virulence (tested on various plant species) of the mutants. However, if the T_R region together with a small part of the adjacent T_L region, which is always present as T_L-DNA in normal crown gall tissues, was deleted the mutants became weakly virulent on Kalanchoë and Nicotiana rustica and avirulent on tomato. We hypothesize that in this case a region necessary for T-DNA integration has been deleted. The same region was found to be essential for lysopine dehydrogenase activity in the tumors.     

Enhancement of the Potential To Utilize Octopine in the Nonfluorescent Pseudomonas sp. Strain 92

The nonfluorescent Pseudomonas sp. strain 92 requires the presence of a supplementary carbon source for growth on octopine, whereas the spontaneous mutant RB100 has acquired the capacity to utilize this opine as the sole carbon and nitrogen source. Insertional mutagenesis of RB100 with transposon Tn5 generated mutants which were unable to grow on octopine and others which grew slowly on this substrate. Both types of mutants yielded revertants that had regained the ability to utilize octopine. Some of the revertants had lost the transposon, whereas in others the transposon was retained but with rearrangements of the insertion site. Genes of octopine catabolism from strain 92 were cloned on a cosmid vector to generate pK3. The clone pK3 conferred the ability to utilize octopine as the sole carbon and nitrogen source on the host Pseudomonas putida KT2440. Although they conferred an equivalent growth phenotype, the mutant genes carried by RB100 and the cloned genes on pK3 differed in their regulation. Utilization of [¹⁴C]octopine was inducible by octopine in RB100 and was constitutive in KT2440(pK3).     

Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens

Summary Several nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed.The plasmid from a nopaline degrading strain has been transferred to a naturally non oncogenic Agrobacterium namely A. radiobacter. Furthermore, the plasmid from an octopine degrading strain has been transferred to a plasmid-cured strain which originally had the capacity to utilize nopaline. Both kinds of experiments prove that the TI plasmid determines the strain specificity with regard to the utilization of either octopine or nopaline.They also demonstrate that the synthesis of either octopine or nopaline in crown gall cells is also determined by genes located on the TI plasmid harboured by the transforming A. tumefaciens strains.     

Octopine as a marker for the induction of tumorous growth by agrobacterium tumefaciens strain B6.

Octopine [N²-(D-1-carboxyethyl)-L-arginine] was detected in all tobacco and sunflower crown gall tumors incited by $\text{Agrobacterium tumefaciens}$ (E. F. Sm. and Town.) Conn strain B₆ at levels between 1 and 2.5 μmoles/20 g fresh weight. Most tissue cultures derived from plant tumors contained octopine at levels between 0.3 and 1 μmole/20 g fresh wt. Normal plant tissues and tissue cultures derived from normal tissues contained no detectable octopine when assayed by a [³H] arginine incorporation technique designed to detect low levels of octopine (less than 0.5 nmole/20 g fresh wt).     

Insights into the Mechanism of Ligand Binding to Octopine Dehydrogenase from Pecten maximus by NMR and Crystallography

Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus, catalyzes the NADH dependent, reductive condensation of L-arginine and pyruvate to octopine, NAD⁺, and water during escape swimming and/or subsequent recovery. The structure of OcDH was recently solved and a reaction mechanism was proposed which implied an ordered binding of NADH, L-arginine and finally pyruvate. Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures. Furthermore, the crystal structure of the OcDH/NADH/agmatine complex was determined which suggests a key role of the side chain of L-arginine in protein cataylsis. Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail.     

Growth of Octopine-Catabolizing Pseudomonas spp. under Octopine Limitation in Chemostats and Their Potential To Compete with Agrobacterium tumefaciens

The growth characteristics of five octopine-catabolizing pseudomonads have been determined in batch and continuous cultures. All five strains belonged to rRNA homology group I and showed a more psychrotrophic growth pattern than did Agrobacterium tumefaciens B6 and ATCC 15955. In chemostats limited by octopine, either as the source of carbon and nitrogen or the sole source of nitrogen, maximum specific growth rates and substrate affinities were lower than those in chemostats limited by glutamate. These growth dynamics were similar to those observed for Agrobacterium strains B6 and ATCC 15955 even though the catabolic genes and pathways are believed to be different in the two genera. An analysis of the yields in octopine-limited chemostats indicated that the use of octopine as the sole source of carbon and nitrogen was grossly inefficient. Octopine and presumably lysopine and octopinic acid provided a better source of nitrogen than of carbon. One of the Pseudomonas fluorescens strains, E175D, was able to produce its highest yield on octopine as a nitrogen source. Competition models formulated on pure culture parameters indicated that two of the Pseudomonas spp. would dominate A. tumefaciens B6 and ATCC 15955 when in simple competition for octopine as a limiting substrate.     

Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: molecular analysis, relationship, and functional characterization.

The occ and noc regions of pTiAch5 (octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The octopine oxidase had high activity with octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.     

Transformation of Brassica napus L. with a 'Disarmed' Octopine Plasmid of Agrobacterium tumefaciens: Molecular Analysis and Inheritance of the Transformed Phenotype

Phenotypically normal and fertile transgenic Brassica napuscv. Westar plants were obtained following co-cultivation ofstem epidermal explants with an Agrobacterium tumefaciens straincontaining a disarmed octopine tumour-inducing plasmid pTiB6S3-SE.The A. tumefaciens cells also contained pMON316, a cointegratevector carrying genes for kanamycin resistance and a scorablemarker nopaline synthase. Transformants were selected by theirability to grow in the presence of 100 µg cm^-3 kanamycin.Transformation was confirmed by the activities of neomycin phosphotransferaseII and nopaline synthase enzymes and by Southern blots. Thekanamycin resistance trait was transferred to the progeny ofthe self-fertilized plants. Key words: Transformation, octopine Ti-plasmid, oilseed rape