Novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one derivatives as potential inhibitors of chorismate mutase

A series of novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one (DHPM) derivatives were designed, synthesized and evaluated in vitro as potential inhibitors of chorismate mutase (CM). All these compounds were prepared via a multi-component reaction (MCR) involving sequential I₂-mediated Biginelli reaction followed by Cu-free Sonogashira coupling. Some of them showed promising inhibitory activities when tested at 30 μM. One compound showed dose dependent inhibition of CM with IC₅₀ value of 14.76 ± 0.54 μM indicating o-alkynylphenyl substituted DHPM as a new scaffold for the discovery of promising inhibitors of CM.     

Molecular cloning and characterization of ARO7-OSM2, a single yeast gene necessary for chorismate mutase activity and growth in hypertonic medium

Summary The chorismate mutase structural gene, ARO7, which is necessary for both phenylalanine and tyrosine biosynthesis was cloned by complementation in yeast. Genetic analysis showed that ARO7 was identical to a gene necessary for growth in hypertonic medium, OSM2, which mapped nearby. After restriction mapping and subcloning of the plasmid, the cloned gene was used to detect mRNA levels in several growth conditions. Enzyme activities were measured in various genotypes. At our level of detection ARO7-OSM2 is a low level constitutively expressed gene.     

Altered control of chorismate mutase leads to tryptophan auxotrophy of Pichia guilliermondii

We have isolated the tryptophan auxotrophic mutant strain, PK101, of Pichia guilliermondii. This strain is not defective in any of the tryptophan biosynthetic enzymes, but its chrismate mutase, an enzyme of the phenylalanine-tyrosine biosynthesis, is changed. In comparison with the wild type chorismate mutase, the enzyme of PK101 is characterized by a complete loss of sensitivity to l-phenylalanine inhibition and to a considerable loss of sensitivity to l-tryptophan activation. Furthermore, the chorismate mutase activity of the mutant is more than 7-fold higher in the absence of l-tryptophan than in the wild type. The PK101 enzyme is also changed in the pH optimum and in some kinetic constants. We found an increased intracellular pool of both phenylalanine and tyrosine and a reduced contents of tryptophan in the mutant cells. Our genetic data indicate that the mutant phenotype is dominant over the wild type.     

Synthesis of 3-indolylmethyl substituted (pyrazolo/benzo)triazinone derivatives under Pd/Cu-catalysis: Identification of potent inhibitors of chorismate mutase (CM)

The chorismate mutase (CM) is considered as an attractive target for the identification of potential antitubercular agents due to its absence in animals but not in bacteria. A series of 3-indolylmethyl substituted pyrazolotriazinone derivatives were designed and docked into CM in silico as potential inhibitors. These compounds were efficiently synthesized using the Pd/Cu-catalyzed coupling-cyclization in a single pot involving the construction of indole ring. The methodology was later extended to the preparation of corresponding benzo analogs of pyrazolotriazinones i.e. 3-indolylmethyl substituted benzotriazinone derivatives. Several of these novel compounds showed significant inhibition of CM when tested in vitro at 30 µM. The SAR (Structure-Activity-Relationship) studies suggested that benzotriazinone moiety was more favorable over the pyrazolotriazinone ring. The two best active compounds showed IC₅₀ ∼ 0.4–0.9 µM (better than the reference/known compounds used) and no toxicity till 30 µM in vitro.     

Electrostatics,allostery, and activity of the yeast chorismate mutase

The predicted active site of chorismate mutase of baker's yeast Saccharomyces cerevisiae has been studied by continuum electrostatics, molecular surface/volume calculations, and molecular modeling. Our study shows that despite being subject to an allosteric transition, the enzyme's active-site pocket neither decreased in volume nor deformed significantly in shape between the active R state and the inactive T state. We find that the polar atmosphere in the pocket is responsible for the enzyme's affinity. A single amino acid, Glu23, can adequately account for the atmospheric variation. This residue swings into the active-site pocket from the R state to the T state. In the R state, Glu23 on helix H2 doubly pairs with Arg204 and Lys208 of H11, which is packed against H2. In the T state, a slide occurs between H11 and H2 such that Glu23 can no longer interact with Lys208 and competes with Asp24 for interacting with Arg204. Consequently, Glu23 is found in the T state to couple with Arg157, an active-site residue critical to substrate binding. The tandem sliding of H11 in both monomers profoundly changes the interactions in the dimer interface. The loop between H11 and H12 demonstrates the largest conformational change. Hence, we establish a connection between the allosteric transition and the activity of the enzyme. The conformational change in the transition is suggested to propagate into the active-site pocket via a series of polar interactions that result in polarity reversal in the active-site pocket, which regulates the enzyme's activity. Proteins 31:445–452, 1998. © 1998 Wiley-Liss, Inc.     

A kinetic model describes metabolic response to perturbations and distribution of flux control in the benzenoid network of Petunia hybrida

In recent years there has been much interest in the genetic enhancement of plant metabolism; however, attempts at genetic modification are often unsuccessful due to an incomplete understanding of network dynamics and their regulatory properties. Kinetic modeling of plant metabolic networks can provide predictive information on network control and response to genetic perturbations, which allow estimation of flux at any concentration of intermediate or enzyme in the system. In this research, a kinetic model of the benzenoid network was developed to simulate whole network responses to different concentrations of supplied phenylalanine (Phe) in petunia flowers and capture flux redistributions caused by genetic manipulations. Kinetic parameters were obtained by network decomposition and non‐linear least squares optimization of data from petunia flowers supplied with either 75 or 150 mm ²H₅‐Phe. A single set of kinetic parameters simultaneously accommodated labeling and pool size data obtained for all endogenous and emitted volatiles at the two concentrations of supplied ²H₅‐Phe. The generated kinetic model was validated using flowers from transgenic petunia plants in which benzyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (BPBT) was down‐regulated via RNAi. The determined in vivo kinetic parameters were used for metabolic control analysis, in which flux control coefficients were calculated for fluxes around the key branch point at Phe and revealed that phenylacetaldehyde synthase activity is the primary controlling factor for the phenylacetaldehyde branch of the benzenoid network. In contrast, control of flux through the β‐oxidative and non‐β‐oxidative pathways is highly distributed.     

Production of chorismate mutase-prephenate dehydrogenase by a strain of Escherichia coli carrying a multicopy,tyrA plasmid: Isolation and properties of the enzyme

A multicopy plasmid that contains the tyrosine operon has been used to transform strains of Escherichia coli K-12. The resultant strains yielded levels of chorismate mutase-prephenate dehydrogenase that were up to 5000-fold higher than that given by the parent strain and about 6-fold higher than that given by a tyrR strain. The production of enzyme fell when tetracycline was omitted from the growth medium because of the loss of the plasmid. The bifunctional enzyme was isolated in good yield by a simple purification procedure and shown to possess properties identical to those exhibited by the enzyme from a tyrR strain.     

1.6 A crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis: novel fold topology revealed

The presence of exported chorismate mutases produced by certain organisms such as Mycobacterium tuberculosis has been shown to correlate with their pathogenicity. As such, these proteins comprise a new group of promising selective drug targets. Here, we report the high-resolution crystal structure of the secreted dimeric chorismate mutase from M. tuberculosis (*MtCM; encoded by Rv1885c), which represents the first 3D-structure of a member of this chorismate mutase family, termed the AroQ(gamma) subclass. Structures are presented both for the unliganded enzyme and for a complex with a transition state analog. The protomer fold resembles the structurally characterized (dimeric) Escherichia coli chorismate mutase domain, but exhibits a new topology, with helix H4 of *MtCM carrying the catalytic site residue missing in the shortened helix H1. Furthermore, the structure of each *MtCM protomer is significantly more compact and only harbors one active site pocket, which is formed entirely by one polypeptide chain. Apart from the structural model, we present evidence as to how the substrate may enter the active site.     

Generation of Phenylpropanoid Pathway-Derived Volatiles in Transgenic Plants: Rose Alcohol Acetyltransferase Produces Phenylethyl Acetate and Benzyl Acetate in Petunia Flowers

Esters are important contributors to the aroma of numerous flowers and fruits. Acetate esters such as geranyl acetate, phenylethyl acetate and benzyl acetate are generated as a result of the action of alcohol acetyltransferases (AATs). Numerous homologous AATs from various plants have been characterized using in-vitro assays. To study the function of rose alcohol acetyltransferase (RhAAT) in planta, we generated transgenic petunia plants expressing the rose gene under the control of a CaMV-35S promoter. Although the preferred substrate of RhAAT in vitro is geraniol, in transgenic petunia flowers, it used phenylethyl alcohol and benzyl alcohol to produce the corresponding acetate esters, not generated by control flowers. The level of benzyl alcohol emitted by the flowers of different transgenic lines was ca. three times higher than that of phenylethyl alcohol, which corresponded to the ratio between the respective products, i.e. ca. three times more benzyl acetate than phenylethyl acetate. Feeding of transgenic petunia tissues with geraniol or octanol led to the production of their respective acetates, suggesting the dependence of volatile production on substrate availability.     

Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol

Petunia flower petals emit large amounts of isoeugenol, which has been shown to be synthesized by isoeugenol synthase (PhIGS1) from an ester of coniferyl alcohol, hypothesized to be coniferyl acetate. This paper describes the identification and characterization of a novel petunia gene encoding an enzyme belonging to the BAHD acyltransferase family whose expression correlates with isoeugenol biosynthesis. RNAi suppression of this gene results in inhibition of isoeugenol biosynthesis. Biochemical characterization of the protein encoded by this gene showed that it has acetyltransferase activity and is most efficient with coniferyl alcohol among the alcohol substrates tested. Overall, these data support the conclusion that coniferyl acetate is the substrate of isoeugenol synthase.     

Development of lady beetle attractants from floral volatiles and other semiochemicals for the biological control of aphids

Aphids are among the most destructive phytophagous pests of host plants, because of their rapid reproduction, parthenogenesis, extensive crop damage, and the transmission of many plant viruses. Since lady beetles are important predatory natural enemies of aphids, developing lady beetle attractants to increase their field abundance is vital for aphid control. Floral volatiles and other semiochemicals are reportedly attractive to lady beetles. In this research, a total of 58 floral volatiles were tested by Y-tube olfactometer assays, among which 29 were highly attractive to both Harmonia axyridis (Coleoptera: Coccinellidae) and Coccinella septempunctata (Coleoptera: Coccinellidae). Meanwhile, the results of wind tunnel trials showed that only isoamyl acetate, α-humulene, trans-3-hexen-1-ol, methyl salicylate, and β-pinene lure these two species. Thereafter, 15 semiochemicals from pests, natural enemies, and pest-infested crops were mixed with the selected floral volatiles, to determine optimum formulations for attracting lady beetles through wind tunnels and further field trials. Eventually, formulas (1) α-humulene: β-pinene: methyl salicylate: trans-3-hexen-1-ol = 1:3:3:10; (5) α-humulene: β-pinene: methyl salicylate:1-nonanal = 1:3:3:10; (8) α-humulene: β-pinene: methyl salicylate: indole = 1:3:3:10 (1, 5, and 8 are labels used for the various formulations in field trials) were successfully verified to be attractive to lady beetles in both pumpkin and wheat fields. These formulations will be of great significance in developing integrated pest management strategies for aphid control.     

Cycad cone and angiosperm floral volatiles: Inferences for the evolution of insect pollination

The chemistry of the volatile emissions of male cones of four putatively insect-pollinated cycad (Cycadales) species and cones of both sexes of one wind-pollinated cycad species was analysed using GC-MS Zamia furfuracea, Macrozamia moorei, and Encephalartos altensteinii had blends consisting primarily of monoterpenoids, benzenoids, and in two causes, a di-unsaturated aliphatic hydrocarbon, while that of Zamia pumila was dominated by methyl salicylate, with sesquiterpenes and aliphatic hydrocarbons also present. In contrast, the wind-pollinated Cycas rumphii contained a series of highly alcohols, ketones and esters. The compounds classes found in the cycads are potent herbivore deterrents, and there is no apparent difference in this regard between insect- and wind-pollinated species. The results suggest convergent evolution in the gymnospermous cycads and magnoliid angiosperms of the olfactory cues that attract pollinating insects; the data are consistent with the hypothesis that early chemical attractants for pollinators evolved from herbivore deterrents.     

A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway

Chorismate synthase, the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants. The enzyme has an absolute requirement for reduced FMN as a cofactor, although the 1,4-anti elimination of phosphate and the C(6proR)-hydrogen does not involve a net redox change. The role of the reduced FMN in catalysis has long been elusive. However, recent detailed kinetic and bioorganic approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an initial electron transfer from tightly bound reduced flavin to the substrate, a process which results in C—O bond cleavage. Studies on chorismate synthases from bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in different ways to chorismate synthase: chorismate synthases in fungi – in contrast to those in bacteria and plants – carry a second enzymatic activity which enables them to reduce FMN at the expense of NADPH. Yet, as shown by the analysis of the corresponding genes, all chorismate synthases are derived from a common ancestor. However, several issues revolving around the origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability of reduced FMN, remain poorly understood. This review summarizes recent developments in the biochemical and genetic arena and identifies future aims in this field. Received: 22 June 1998 / Accepted: 7 August 1998