C-N bond formation under Cu-catalysis: Synthesis and in vitro evaluation of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones against chorismate mutase

A series of novel N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones were designed and synthesized as potential inhibitors of chorismate mutase. Synthesis of this class of compounds was carried out by using Cu-mediated C-N bond forming reaction between thieno[2,3-d]pyrimidin-4(3H)-ones and aryl boronic acids. The reaction can be performed in an open flask as the conversion was found to be not sensitive to the presence of air or atmospheric moisture. A range of compounds were prepared by using this method and single crystal X-ray diffraction study was performed using a representative compound. In vitro pharmacological data of some of the compounds synthesized along with dose response studies using active molecules are presented. In silico interactions of these molecules with chorismate mutase are also presented.     

Absolute dependence of phenylalanine and tyrosine biosynthetic enzyme on tryptophan in Candida maltosa

Candida maltosa synthesizes phenylalanine and tyrosine only via phenylpyruvate and p-hydroxyphenylpyruvate. Tryptophan is absolutely necessary for the enzymatic reaction of chorismate mutase and prephenate dehydrogenase; activity of prephenate dehydratase can be increased 2.5-fold in the presence of tryptophan. Activation of the chorismate mutase, prephenate dehydratase and prephenate dehydrogenase by tryptophan is competitive with respect to chorismate and prephenate with Ka 0.06mM, 0.56mM and 1.7mM. In addition tyrosine is a competitive inhibitor of chorismate mutase (Ki = 0.55mM) and prephenate dehydrogenase (Ki = 5.5mM).     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.     

Chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes: evidence that the two reactions occur at one active site.

The relationship between the sites for catalysis of two reactions by the bifunctional enzyme chorismate mutase--prephenate dehydrogenase has been investigated. The results are consistent with the occurrence of both reactions at one active site. Comparisons have been made between experimental data for the time course of the overall reaction and computer simulations, according to various models for the relationship between the mutase and dehydrogenase sites. A model based on a single active site is consistent with the time course data if a minor proportion of the chorismate that reacts can be converted through to (hydroxyphenyl)pyruvate without the intermediate release of prephenate. Consistent with this requirement, some channeling of radioactivity from chorismate to (hydroxyphenyl)pyruvate has been detected. A model based on two separate sites has also been considered; the simulations show that if this model applies there is no need to postulate any channeling of the intermediate, prephenate, between the sites and there must be marked inhibition of the dehydrogenase reaction by chorismate. Since channeling has been observed and chorismate increases the dehydrogenase rate under all conditions, the two-site model appears unlikely. Consistent with the one-site model are the observations that a variety of inactivating conditions cause parallel loss of mutase and dehydrogenase activity and that identical protection against inactivation of both mutase and dehydrogenase by iodoacetamide is afforded by prephenate.     

Degradation and biosynthesis of L-phenylalanine by chloridazon-degrading bacteria]

Incubating chloridazon-degrading bacteria with L-phenylalanine leads to the accumulation of L-2,3-dihydroxyphenylalanine, o-tyrosine and m-tyrosine in the medium. Incubating the bacteria with N-acetyl-L-phenylalanine leads to N-acetyl-(2,3-dihydroxyphenyl)alanine. Using phenylacetic acid as substrate leads to the accumulation of malonic acid. The products are isolated by gel chromatography and high performance liquid chromatography. 2,3-Dihydroxy-L-phenylalanine is attacked by a catechol 2,3-dioxygenase in the presence of Fe2. An unstable yellow compound is formed in this reaction. This meta-cleavage-product is again cleaved by a hydrolase, leading to aspartic acid and 4-hydroxy-2-oxovaleric acid. Both products were isolated fromthe reaction buffer by amino acid analysis and high performance liquid chromatography. The dioxygenase and hydrolase were partially purified and characterized. A new degradation pathway for phenylalanine is discussed and compared with known pathways. The enzymes chorismate mutase, prephenate dehydratase and prephenate dehydrogenase are characterized and inhibition as well as repression are investigated. Only prephenate dehydrogenase is inhibited by phenylalanine, tyrosine and tryptophane. Chorismate mutase is repressed by phenylalanine, prephenate dehydrogenase by phenylalanine and tyrosine. Prephenate dehydratase is not repressed by aromatic amino acids. Regulation of aromatic amino acid biosynthesis in connection with phenylalanine degradation is discussed.     

Comparison of chorismate mutase isozyme patterns in selected plants

A wide variety of plants have been assayed to determine if they contain three isozymes of chorismate mutase (EC 5.4.99.5) as does alfalfa (Medicago sativa L.) or two isozymes, as does mung bean (Phaseolus aureus). The isozymes were separated by disc electrophoresis. All anthophyta with the exception of some closely related Leguminosae contained three isozymes of chorismate mutase. The one coniferophyta (a pine), and pterophyta (a fern) and one microphyllophyta (a Selaginella) assayed contained two isozymes of chorismate mutase. All plants assayed contained measurable chorismate mutase levels and at least two isozymes of chorismate mutase.     

A Pd-mediated new strategy to functionalized 2-aminochromenes: their in vitro evaluation as potential anti tuberculosis agents

A multi component based synthesis involving palladium catalyzed C-C bond forming reaction has been developed as a new strategy to access systematically modified functionalized 2-aminochromenes. This MCR involves the use of bromobenzaldehyde as a key component and is highlighted by generating a new compound library. Many of these compounds showed Mycobacterium tuberculosis H37Rv chorismate mutase inhibiting properties in vitro representing the lead example of chorismate mutase inhibition by heteroarene based compounds.     

Mapping Enzymatic Catalysis Using the Effective Fragment Molecular Orbital Method: Towards all ab initio Biochemistry

We extend the Effective Fragment Molecular Orbital (EFMO) method to the frozen domain approach where only the geometry of an active part is optimized, while the many-body polarization effects are considered for the whole system. The new approach efficiently mapped out the entire reaction path of chorismate mutase in less than four days using 80 cores on 20 nodes, where the whole system containing 2398 atoms is treated in the ab initio fashion without using any force fields. The reaction path is constructed automatically with the only assumption of defining the reaction coordinate a priori. We determine the reaction barrier of chorismate mutase to be kcal mol⁻¹ for MP2/cc-pVDZ and for MP2/cc-pVTZ in an ONIOM approach using EFMO-RHF/6-31G(d) for the high and low layers, respectively.     

Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity

Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.     

Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non‐native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues.     

Chorismate mutase-prephenate dehydratase from Escherichia coli: active sites of a bifunctional enzyme.

The relationship between the active sites of the bifunctional enzyme chorismate mutase-prephenate dehydratase has been examined. Steady-state kinetic investigations of the reactions with chorismate or prephenate as substrate and studies of the overall conversion of chorismate to phenylpyruvate indicate that there are two distinct active sites. One site is responsible for the mutase activity and the other for the dehydratase activity. Studies of the overall reaction using radioactive chorismate show that prephenate, which is formed from chorismate, dissociates from the mutase site and equilibrates with the bulk medium before combining at the dehydratase site. No evidence was obtained for direct channeling of prephenate from one site to the other, or for any strong interaction between the sites.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.     

Regulated enzymes of aromatic amino acid synthesis: control, isozymic nature, and aggregation in Bacillus subtilis and Bacillus licheniformis

Several regulated enzymes involved in aromatic amino acid synthesis were studied in Bacillus subtilis and B. licheniformis with reference to organization and control mechanisms. B. subtilis has been previously shown (23) to have a single 3-deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase but to have two isozymic forms of both chorismate mutase and shikimate kinase. Extracts of B. licheniformis chromatographed on diethylaminoethyl (DEAE) cellulose indicated a single DAHP synthetase and two isozymic forms of chorismate mutase, but only a single shikimate kinase activity. The evidence for isozymes has been supported by the inability to find strains mutant in these activities, although strains mutant for the other activities were readily obtained. DAHP synthetase, one of the isozymes of chorismate mutase, and one of the isozymes of shikimate kinase were found in a single complex in B. subtilis. No such complex could be detected in B. licheniformis. DAHP synthetase and shikimate kinase from B. subtilis were feedback-inhibited by chorismate and prephenate. DAHP synthetase from B. licheniformis was also feedback-inhibited by these two intermediates, but shikimate kinase was inhibited only by chorismate. When the cells were grown in limiting tyrosine, the DAHP synthetase, chorismate mutase, and shikimate kinase activities of B. subtilis were derepressed in parallel, but only DAHP synthetase and chorismate mutase were derepressible in B. licheniformis. Implications of the differences as well as the similarities between the control and the pattern of enzyme aggregation in the two related species of bacilli were discussed.     

Cu-mediated N-arylation of 1,2,3-triazin-4-ones: synthesis of fused triazinone derivatives as potential inhibitors of chorismate mutase

A rapid and direct access to N-aryl substituted fused triazinone derivatives has been accomplished via N-arylation of 1,2,3-triazin-4-one ring involving a Cu-mediated coupling between triazinone derivatives and aryl boronic acids. A combination of Cu(OAc)(2)-Et(3)N in 1,2-dichloroethane was found to be effective and various fused triazinone derivatives have been prepared by using this methodology. Molecular structure of a representative compound was confirmed by single crystal X-ray diffraction study. The scope and limitations of this reaction is discussed. Some of the compounds synthesized were tested for chorismate mutase inhibitory properties in vitro. The in vitro dose response study of an active compound is presented.     

Increased Chorismate Mutase Levels as a Response to Wounding in Solanum tuberosum L. Tubers

Discs excised from Solanum tuberosum L. cv White Rose tubers demonstrated a 4.5-fold increase in chorismate mutase activity 48 hours after excision. Incubation in the presence of cycloheximide (25 micromolar) or actinomycin D (100 micromolar) completely inhibited the wound response suggesting de novo synthesis of chorismate mutase. Ratios of activity in the presence of the activator tryptophan to that in the absence of tryptophan remained relatively constant during the induction period. This indicated either a constant ratio of tryptophan sensitive to tryptophan insensitive isozymes, or that only one form of chorismate mutase was present. Chromatography of crude extracts on three different columns yielded only one peak of chorismate mutase activity, activated by tryptophan in each case. Incubation under white light had no effect on chorismate mutase activity when compared to dark controls.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: positive cooperativity with substrates and inhibitors

Investigations have been made at pH 6.0 of the effect of chorismate and adamantane derivatives on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase from Escherichia coli. When used over a wide range of concentrations, chorismate 5,6-epoxide, chorismate 5,6-diol, adamantane-1,3-diacetate, adamantane-1-acetate, adamantane-1-carboxylate, and adamantane-1-phosphonate give rise to nonlinear plots of the reciprocal of the initial velocity of each reaction as a function of the inhibitor concentration. The inhibitors do not induce the enzyme to undergo polymerization and have only a small effect on the S20,w value of the enzyme as determined by using sucrose density gradient centrifugation. At low substrate concentration, low concentrations of adamantane-1-acetate cause activation of both the mutase and dehydrogenase activities while at higher concentrations this compound functions as an inhibitor. When chorismate and prephenate are varied over a wide range of concentrations, double-reciprocal plots of the data indicate that the reactions exhibit positive cooperativity. The addition of albumin eliminates the cooperative interactions associated with substrates but has little effect on those associated with inhibitors.     

Relationships Among Mycobacteria and Nocardiae Based upon Deoxyribonucleic Acid Reassociation

Chorismate mutase from Streptomyces aureofaciens was purified 12-fold. This enzyme preparation did not show any activity when tested for anthranilate synthetase, prephenate dehydrogenase, or prephenate dehydratase. The catalytic activity of chorismate mutase has a broad optimum between pH 7 and 8. The initial velocity data followed regular Michaelis-Menten kinetics with a K(m) of 5.3 x 10(-4) M, and the molecular weight of the enzyme was determined by sucrose gradient centrifugation to be 50,000. Heat inactivation of chorismate mutase, which occurs above temperatures of 60 C, is reversible. The enzyme activity can be restored even when chorismate mutase is treated at the temperature of a boiling-water bath for 15 min. Heat-denatured and renatured enzymes showed the same Michaelis constant and the same molecular weight as the native enzyme. l-Phenylalanine, l-tyrosine, l-tryptophan, and metabolites of the aromatic amino acid pathway were tested as potential modifiers of chorismate mutase activity. The activity of the enzyme was inhibited by none of these substances. Chorismate mutase of S. aureofaciens was not repressed in cells grown in minimal medium supplemented with l-phenylalanine, l-tyrosine, or l-tryptophan.     

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K_a for chorismate and the K_is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.     

Ligand based virtual screening and biological evaluation of inhibitors of chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv

We have identified new lead candidates that possess inhibitory activity against Mycobacterium tuberculosis H37Rv chorismate mutase by a ligand-based virtual screening optimized for lead evaluation in combination with in vitro enzymatic assay. The initial virtual screening using a ligand-based pharmacophore model identified 95 compounds from an in-house small molecule database of 15,452 compounds. The obtained hits were further evaluated by molecular docking and 15 compounds were short listed based on docking scores and the other scoring functions and subjected to biological assay. Chorismate mutase activity assays identified four compounds as inhibitors of M. tuberculosis chorismate mutase (MtCM) with low K(i) values. The structural models for these ligands in the chorismate mutase binding site will facilitate medicinal chemistry efforts for lead optimization against this protein.     

Monofunctional chorismate mutase from Bacillus subtilis: kinetic and 13C NMR studies on the interactions of the enzyme with its ligands

The interaction of the monofunctional chorismate mutase from Bacillus subtilis with chorismate and prephenate has been studied kinetically and by NMR spectroscopy with 13C specifically labeled substrates. Prephenate dominates the population of enzyme-bound species, and the "off" rate constant (approximately 60 s-1) obtained from line-broadening experiments is close to the value of kcat for chorismate (50 s-1) determined kinetically. The calculated "on" rate constant for prephenate (8 x 10(5) M-1 s-1) is similar to the value of kcat/Km for chorismate (5 x 10(5) M-1 s-1). The kinetic parameters of the Bacillus mutase are remarkably insensitive to pH over a wide range and display no solvent isotope effect. These results suggest that the enzyme-catalyzed reaction may be encounter controlled (slowed from the diffusion limit by some feature of the enzyme's active site) and that kcat for chorismate is determined by the product off rate. There is now no evidence to suggest that the skeletal rearrangement on the enzyme surface occurs by a pathway other than a pericyclic process.