Isopentenyl Group Research Articles

Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases.

It has been believed that the key step in cytokinin biosynthesis is the addition of a 5-carbon chain to the N(6) of AMP. To identify cytokinin biosynthesis enzymes that catalyze the formation of the isopentenyl side chain of cytokinins, the Arabidopsis genomic sequence was searched for genes that could code for isopentenyltransferases. This resulted in the identification of nine putative genes for isopentenyltransferases. One of these, AtIPT4, was subjected to detailed analysis. Overexpression of AtIPT4 caused cytokinin-independent shoot formation on calli. As shoot formation on calli normally occurs only when cytokinins are applied, it suggested that this gene product catalyzed cytokinin biosynthesis in plants. Recombinant AtIPT4 catalyzed the transfer of an isopentenyl group from dimethylallyl diphosphate to the N(6) of ATP and ADP, but not to that of AMP. AtIPT4 did not exhibit the DMAPP:tRNA isopentenyltransferase activity. These results indicate that cytokinins are, at least in part, synthesized from ATP and ADP in plants.

Prenylflavonoids: A New Class of Non-Steroidal Phytoestrogen (Part 1). Isolation of 8-Isopentenylnaringenin and an Initial Study on its Structure-Activity Relationship

Bioassay-guided fractionation of a methanolic extract of a Thai crude drug, derived from heartwood of Anaxagorea luzonensis A. Gray (Annonaceae), resulted in the isolation of 8-isopentenylnaringenin (1) as an estrogen agonist with a activity of about an order of magnitude greater than genistein. Various flavonoids possessing isopentenyl side chains in the A-ring have been prepared and evaluated for their ability to bind estrogen receptor. In addition, enantiomers of 1 were separated and the respective enantiomers were assayed. These studies have demonstrated that the presence of an 8-isopentenyl group is an important factor for binding. Flavones, flavanones and flavonols having an isopentenyl substituent at C-8 exhibited an appreciable affinity for estrogen receptor. Conversely, isoflavones possessing an 8-isopentenyl substituent at C-8 did not show this activity. Movement of the isopentenyl group from position 8 to 6 resulted in loss of the activity. No significant difference was observed between 2(S)- and 2(R)-enantiomers of 1 in their binding affinity. Prenylflavonoids are reported to possess a wide range of biological activities; however, estrogenic activity has not been described.

The ms2io6A37 modification of tRNA in Salmonella typhimurium regulates growth on citric acid cycle intermediates.

The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present in position 37 (adjacent to and 3' of the anticodon) of tRNAs that read codons beginning with U except tRNA(i.v. Ser) in Escherichia coli. In Salmonella typhimurium, 2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine (ms2io6A; also referred to as 2-methylthio cis-ribozeatin) is found in tRNA, most likely in the species that have ms2i6A in E. coli. Mutants (miaE) of S. typhimurium in which ms2i6A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of S. typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms2io6A37 and is not due to a second cellular function of tRNA (ms2io6A37)hydroxylase, the miaE gene product. We suggest that S. typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.

Host mutations (miaA and rpsL) reduce tetracycline resistance mediated by Tet(O) and Tet(M).

The effects of mutations in host genes on tetracycline resistance mediated by the Tet(O) and Tet(M) ribosomal protection proteins, which originated in Campylobacter spp. and Streptococcus spp., respectively, were investigated by using mutants of Salmonella typhimurium and Escherichia coli. The miaA, miaB, and miaAB double mutants of S. typhimurium specify enzymes for tRNA modification at the adenosine at position 37, adjacent to the anticodon in tRNA. In S. typhimurium, this involves biosynthesis of N6-(4-hydroxyisopentenyl)-2-methylthio-adenosine (ms2io6A). The miaA mutation reduced the level of tetracycline resistance mediated by both Tet(O) and Tet(M), but the latter showed a greater effect, which was ascribed to the isopentenyl (i6) group or to a combination of the methylthioadenosine (ms2) and i6 groups but not to the ms2 group alone (specified by miaB). In addition, mutations in E. coli rpsL genes, generating both streptomycin-resistant and streptomycin-dependent strains, were also shown to reduce the level of tetracycline resistance mediated by Tet(O) and Tet(M). The single-site amino acid substitutions present in the rpsL mutations were pleiotropic in their effects on tetracycline MICs. These mutants affect translational accuracy and kinetics and suggest that Tet(O) and Tet(M) binding to the ribosome may be reduced or slowed in the E. coli rpsL mutants in which the S12 protein is altered. Data from both the miaA and rpsL mutant studies indicate a possible link between stability of the aminoacyl-tRNA in the ribosomal acceptor site and tetracycline resistance mediated by the ribosomal protection proteins.

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5′‐AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin‐containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N6‐side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.

Conformational preferences of modified nucleic acid bases N6‐(Δ2‐isopentenyl) adenine and 2‐methylthio‐N6‐(Δ2‐isopentenyl) adenine by the quantum chemical PCILO calculations

AbstractConformational preferences of the hypermodified nucleic acid bases N6‐(Δ2‐isopentenyl) adenine, (i6Ade) and its 2‐methylthio derivative (ms2i6Ade) have been investigated theoretically by the quantum chemical perturbative configuration interaction using localized orbitals (PCILO) method.The predicted most stable conformation of i6Ade and ms2i6Ade are such that the isopentenyl substituent is oriented away from the imidazole moiety of the adenine ring. The atoms N(6), C(10), and C(11) remain coplanar with the adenine ring for both molecules. However, in contrast to the predicted cis orientation of the C(10)C(11) bond with respect to the C(6)N(6) bond in i6Ade, the trans orientation is favored for ms2i6Ade. The plane of the isopentenyl group is rotated by 120° from that of the purine base in i6Ade, whereas rotation by 60° is favored in ms2i6Ade. The favored orientation of the methylthio group with respect to the C(2)N(3) bond is trans; however, the alternative cis arrangement is also quite probable. The conformational implications of the methylthiolation of the isopentenyladenine are brought out in the context of the considerably large range of accessible (β,γ) and χ orientations. The compatible roles of i6Ade and ms2i6Ade in tRNA are thus understood, besides their distinguishing features.

Purification and some properties of Δ2-isopentenylpyrophosphate:5′AMP Δ2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum

Δ 2-Isopentenylpyrophosphate:5′AMP Δ 2-isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from Δ 2-isopentenylpyrophosphate and 5′AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5′AMP ox-redAH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 ± 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn 2+, Zn 2+, and Mg 2+ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5′AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrate, being 60–80% as effective as 5′AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 m m Zn 2+, and 25 °C) the K m values for 5′AMP and Δ 2-isopentenylpyrophosphate were 1.0 × 10 −7 m and 2.2 × 10 −6 m, respectively.

Cell-free biosynthesis of cytokinins in cultured tobacco cells

Cell-free biosynthesis of free cytokinins not related to the degradation of t-RNA was demonstrated by using enzyme preparation extracted from cultured tobacco (Nicotiana tabacum cv. Xanthi) cells. The reaction mixture for cytokinin biosynthesis contained 20 mM Tris-HCl buffer (pH 7.6), 5 mM magnesium acetate, 0.5 mM isopentenylpyrophosphate, 50 μM adenosine as a substrate and dialyzed enzyme extract. After 2.5 hours of reaction at 27°C, N6-(Δ2-isopentenyl)adenosine, N6-(Δ2-isopentenyl)adenine and trans-zeatin were biosynthesized. The radioactivity of [U-14C]adenosine was incorporated into these three cytokinins after 30 minutes of reaction in the cell-free system. From the time course of biosynthesis of cytokinins and the incorporation of [U-14C]adenosine into cytokinins, we concluded that adenosine accepts the isopentenyl group directly to form N6-(Δ2-isopentenyl) adenosine. This is converted to trans-zeatin via N6-(Δ2-isopentenyl)adenine by several enzymes.

5'-AMP is a direct precursor of cytokinin in Dictyostelium discoideum.

ISOPENTENYLADENINE (i6Ade) and closely related derivatives occur naturally either as free bases or as constituents of tRNA1,2. Although these cytokinins have been detected in various organisms, it is still not clear whether they are produced by the degradation of tRNA or whether they are synthesised by some pathway not involving tRNA. A spore germination inhibitor, discadenine, isolated from Dictyostelium discoideum was found to be an i6Ade derivative with a 3-amino-3-carboxypropyl group in the 3 position3—the cells contained a large quantity of free i6Ade, presumably a precursor of discadenine4. These results led us to investigate the cell-free biosynthesis of i6Ade in this organism. We report here the existence of a cytokinin biosynthetic pathway not related to tRNA degradation, showing that 5′-AMP is the actual acceptor molecule for the isopentenyl group. Our findings suggest that the biosynthesis of cytokinin in higher plants may proceed in a similar fashion.

Antibodies to N6-(delta2-isopentenyl) adenosine and its nucleotide: interaction with purified tRNAs and with bases, nucleosides and nucleotides of the isopentenyladenosine family.

The interaction of antibodies directed toward N6-(delta2-isopentenyl)adenosine, i6Ado, or its nucleotide with related bases, nucleosides, nucleotides and purified tRNAs is described. The selectivity of the antibody preparation was tested in inhibition experiments utilizing a sensitive radioimmunoassay to quantitate the binding of [3H]i6Ado to the antibody. Purified tRNAs containing various modified nucleosides adjacent to the 3'-end of the anticodon were tested to provide information about the selectivity of the antibody preparation toward nucleotides in this position of the tRNA chain. Antibodies directed against the nucleotide hapten were used to purify tRNAs which contain i6Ado and to quantitate the amount of that nucleotide. The same order of selectivity was expressed whether the nucleotides were free or in a tRNA molecule. Interaction of the antibody with compounds from the i6Ado family demonstrated dominance of the hydrophobic isopentenyl group and the importance of positional differences of modifications.

The reactions of oxo-osmium ligand complexes with isopentenyl adenine and its nucleoside

We report syntheses of oxo-osmium(VI)bis(ligand) esters of N 6(Δ 2isopentenyl) adenine (6-ipAde) and its nucleoside (IPA) which result from the addition of OsO 4 to the double bond of the isopentenyl group. A study of the kinetics of these reactions shows that under typical conditions the rates of reaction relative to thymidine are as follows: for OsO 4-pyridine: thymidine = 1; 6-ipAde = 4600: for OsO 4-2, 2′-bipyridyl: thymidine = 380; 6-ipAde = 8600; IPA = 8600. We also report syntheses of osmate esters of IPA in which the osmium is bonded through the 2′- and 3′-hydroxyl groups of the ribose residue.

The structure of psoralidin

Psoralidin, C 20H 16O 5 the phenolic coumarin of Psoralea corylifolia Linn. has been shown to possess two hydroxyl groups, one conjugated δ-lactone, one isopentenyl group and an intramolecular ether linkage. On systematic degradation dihydropsoralidin dimethyl ether gave 2-hydroxy-4-methoxybenzaldehyde and 2,4-dimethoxy-5-(-3-methylbutyl)-benzoic acid. The synthesis of the latter compound has been described. On the basis of the degradation experiments coupled with ultra-violet and infra-red absorption data, the structure of psoralidin has been established as 6-(3-methylbut-2-enyl)-coumestrol (IV, R = H).