Combinatorial biosynthesis for new drug discovery

Combinatorial biosynthesis involves interchanging secondary metabolism genes between antibiotic-producing microorganisms to create unnatural gene combinations or hybrid genes if only part of a gene is exchanged. Novel metabolites can be made by both approaches, due to the effect of a new enzyme on a metabolic pathway or to the formation of proteins with new enzymatic properties. The method has been particularly successful with polyketide synthase (PKS) genes: derivatives of medically important macrolide antibiotics and unusual polycyclic aromatic compounds have been produced by novel combinations of the type I and type II PKS genes, respectively. Recent extensions of the approach to include deoxysugar biosynthesis genes have expanded the possibilities for making new microbial metabolites and discovering valuable drugs through the genetic engineering of bacteria.     

Do electrostatic interactions determine glycation of hyaluronidase derivatives with N-acetylhexosamines?

Using N-acetylglucosamine and N-acetylgalactosamine as model agents for glycation of native hyaluronidase and its chondroitin sulfate modified form it has been shown that the modified enzyme exhibited higher inactivation than the native enzyme, while heparin caused similar inhibition of both forms. Such effect could be attributed to the development of electrostatic interactions as the modified hyaluronidase had altered surface electrostatic potential after chondroitin sulfate binding. However, variations in ionic strength of the medium containing enzyme derivatives have shown that their endoglycosidase activity changed in a similar manner and the effect on glycation represents a multifactor process. N-acetylhexosamines are natural labels of endothelial glycocalyx degradation products. Interaction of the hyaluronidase forms with charged hyaluronan fragments revealed significantly higher inactivation of the modified enzyme compared with the native enzyme. The glycation pattern observed in this study was opposite to that observed with mono- and disaccharides. Thus, it appears that the investigated hyaluronidase derivatives represent an informative enzymatic test in vivo for determination of the dominant type of glycation agents in blood circulation and their origin.     

Catabolism of thyroliberin by rat adenohypophyseal tissue extract

Rapid fragmentation of thyroliberin (less than Glu-His-Pro-NH2) by rat adenohypophyseal tissue enzymes could be demonstrated. Based on the identification of the metabolic products and by the demonstration that the individual enzymatic reactions can be preferentially blocked by enzyme inhibitors, specific and sensitive biochemical tests could be developed in order to monitor the enzymatic activities after gel chromatographic fractionation of the tissue extracts. These findings are in agreement with the interpretation that the observed degradation of thyroliberin by hypophyseal tissue extracts may follow the proposed pathways. The primary enzymatic cleavage of thyroliberin is either initiated by the action of a 'thyroliberin-deamidating enzyme' (thyroliberin leads to less than Glu-His-Pro-OH + NH3), or by the action of a pyroglutamate aminopeptidase (thyroliberin leads to less than Glu + His-Pro-NH2). While the pyroglutamate aminopeptidase also catalyzes the subsequent degradation of deamidated thyroliberin (less than Glu-His-Pro-OH leads to less than Glu + His-Pro-OH), the enzymatic deamidation of His-Pro-NH2 is not catalyzed by the 'thyroliberin-deamidating enzyme; but by a post-proline dipeptidyl aminopeptidase. Hydrolysis of the common intermediary metabolite His-Pro-OH to the free amino acids is apparently catalyzed by a proline dipeptidase. In addition to these enzymatic events rapid cyclization of His-Pro-NH2 to histidyl-proline-diketopiperazine His-Pro could be observed. This reaction however is mainly due to the non-enzymatic intramolecular condensation reaction which is characteristic for proline-containing dipeptide derivatives. An enzymatic activity which catalyzes this reaction could not be observed when the enzyme fractions were tested. Enzymatic degradation of His-Pro by hypophyseal tissue extracts could also not be observed.     

Enzymatic biotransformation of terpenes as bioactive agents

The plant-derived terpenoids are considered to be the most potent anticancer, anti-inflammatory and anticarcinogenic compounds known. Enzymatic biotransformation is a very useful approach to expand the chemical diversity of natural products. Recent enzymatic biotransformation studies on terpenoids have resulted in the isolation of novel compounds. 14-hydroxy methyl caryophyllene oxide produced from caryophyllene oxide showed a potent inhibitory activity against the butyryl cholinesterase enzyme, and was found to be more potent than parent caryophyllene oxide. The metabolites 3β,7β-dihydroxy-11-oxo-olean-12-en-30-oic acid, betulin, betulonic acid, argentatin A, incanilin, 18β glycyrrhetinic acid, 3,11-dioxo-olean-12-en-30-oic acid produced from 18β glycyrrhetinic acid were screened against the enzyme lipoxygenase. 3,11-Dioxo-olean-12-en-30-oic acid, was found to be more active than the parent compound. The metabolites 3β-hydroxy sclareol 18α-hydroxy sclareol, 6α,18α-dihydroxy sclareol, 11S,18α-dihydroxy sclareol, and 1β-hydroxy sclareol and 11S,18α-dihydroxy sclareol produced from sclareol were screened for antibacterial activity. 1β-Hydroxy sclareol was found to be more active than parent sclareol. There are several reports on natural product enzymatic biotransformation, but few have been conducted on terpenes. This review summarizes the classification, advantages and agents of enzymatic transformation and examines the potential role of new enzymatically transformed terpenoids and their derivatives in the chemoprevention and treatment of other diseases.     

A model for enzymatic isomerization of D-glucose to D-fructose in a batch reactor.

Using whole cells containing glucose isomerase, mathematical models for the enzymatic conversion of D-glucose to D-fructose and for the inactivation of the enzyme catalyst have been postulated and verified experimentally. The heat of reaction, the equilibrium constant, and the individual rate constants and their activation energies have been estimated. The model can be used to predict the time course for the enzymatic production of fructose in a batch reactor within the tested experimental range of 40-80 degrees C.     

Multiple effects of chemical reagent on enzyme: o-phthalaldehyde-induced inactivation,dissociation and partial unfolding of lactate dehydrogenase from pig heart

The effects of o-phthalaldehyde (OPTA) on lactate dehydrogenase (LDH) have been studied by following changes in enzymatic activity, aggregation state and conformation. Treatment with OPTA resulted in pseudo first-order inactivation of LDH over a wide concentration range of the inhibitor, and the second-order rate constant was estimated to be 1.52 M⁻¹ s⁻¹. The loss of enzyme activity was concomitant with the increases in absorbance at 337 nm and fluorescence intensity at 405 nm. Complete loss of enzyme activity was accompanied by the formation of approximately 4 mol isoindole derivatives per mole LDH subunit. Cross-linking experiments verified enzyme dissociation during OPTA modification, which could be attributed to the modification of both thiol groups and lysine residues. Circular dichroism (CD) spectra showed that the secondary structure of the OPTA-modified enzyme decreased correspondingly. Comparison of the inactivation with the conformational changes of the enzyme suggests that the active site of the enzyme exhibits greater conformational flexibility than the enzyme molecule as a whole. It is concluded that OPTA modification has multiple effects on LDH, including its inactivation, dissociation and partial unfolding.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

Drosophila melanogaster dihydroorotate dehydrogenase: the N-terminus is important for biological function in vivo but not for catalytic properties in vitro

Dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis, is an integral flavoprotein of the inner mitchondrial membrane and is functionally connected to the respiratory chain. Here, experiments have been directed toward determining the roles of the N-terminal sequence motifs both in enzymatic properties of insect DHODH produced in vitro and the in vivo function of the protein. Full-length and three N-terminal truncated derivatives of the Drosophila melanogaster enzyme were expressed in Escherichia coli and purified. For identification on Western blots of recombinant DHODH as well as the native enzyme from flies polyclonal anti-DHODH immunoglobulins were generated and affinity-purified. The enzymatic characteristics of the four versions of DHODH were very similar, indicating that the N-terminus of the enzyme does not influence its catalytic function or its susceptibility to prominent DHODH inhibitors: A77-1726, brequinar, dichloroallyl-lawsone and redoxal. Whereas the efficacy of A77-1726 and dichloroallyl-lawsone were similar with Drosophila and human DHODH, that of brequinar and redoxal differed significantly. The differences in responses of insect DHODH and the enzyme from other species may allow the design of new agents that will selectively control insect growth, due to pyrimidine nucleotide limitation. In vivo expression of the full-length and N-truncated DHODHs from engineered transgenes revealed that the truncated proteins could not support normal de novo pyrimidine biosynthesis during development of the fly (i.e., failure to complement dhod-null mutations), apparently due to instability of the truncated proteins. It is concluded that the proper intracellular localization, directed by the N-terminal targeting and transmembrane motifs, is required for stability and subsequent proper biological function in vivo.     

Impact of aryloxy-linked quinazolines: a novel series of selective VEGFR-2 receptor tyrosine kinase inhibitors

Three series of 6,7-dimethoxyquinazoline derivatives substituted in the 4-position by aniline, N-methylaniline and aryloxy entities, targeting EGFR and VEGFR-2 tyrosine kinases, were designed and synthesized. Pharmacological activities of these compounds have been evaluated for their enzymatic inhibition of VEGFR-2 and EGFR and for their antiproliferative activities on various cancer cell lines. We have studied the impact of the variation in the 4-position substitution of the quinazoline core. Substitution by aryloxy groups led to new compounds which are selective inhibitors of VEGFR-2 enzyme with IC₅₀ values in the nanomolar range in vitro.     

Integrating thermodynamic and enzymatic constraints into genome-scale metabolic models

Stoichiometric genome-scale metabolic network models (GEMs) have been widely used to predict metabolic phenotypes. In addition to stoichiometric ratios, other constraints such as enzyme availability and thermodynamic feasibility can also limit the phenotype solution space. Extended GEM models considering either enzymatic or thermodynamic constraints have been shown to improve prediction accuracy. In this paper, we propose a novel method that integrates both enzymatic and thermodynamic constraints in a single Pyomo modeling framework (ETGEMs). We applied this method to construct the EcoETM (E. coli metabolic model with enzymatic and thermodynamic constraints). Using this model, we calculated the optimal pathways for cellular growth and the production of 22 metabolites. When comparing the results with those of iML1515 and models with one of the two constraints, we observed that many thermodynamically unfavorable and/or high enzyme cost pathways were excluded from EcoETM. For example, the synthesis pathway of carbamoyl-phosphate (Cbp) from iML1515 is both thermodynamically unfavorable and enzymatically costly. After introducing the new constraints, the production pathways and yields of several Cbp-derived products (e.g. L-arginine, orotate) calculated using EcoETM were more realistic. The results of this study demonstrate the great application potential of metabolic models with multiple constraints for pathway analysis and phenotype prediction.     

Surface-bound aspartate aminotransferase on collagen films. Compared properties with native enzyme.

Aspartate aminotransferase (L-aspartate : 2-oxoglutarate aminotransferase, EC 2.6.1.1) has been covalently bound to chemically activated collagen films. This enzyme had never previously been coupled to any other solid support. The coupling method, including acyl azide formation on the carrier, allowed coupling of many other enzymes. A systematic study of coupling conditions has been performed; influence of time of coupling and of concentration of coupling solution on the enzymatic activity retained on the film. Coupling solutions could be used for several successive couplings. To determine the yield of binding, N-[14C] ethylmaleimide-labelled enzyme was prepared fully active and bound to collagen films. After lyophilisation the film retained most of its activity when stored in buffer and the half-life of the enzymatic film was about ten months. pH Dependence and activation energy were about the same for soluble and coupled enzyme. Coupling protects against thermal denaturation and increases the stability of the enzyme; the enzymatic film could be used repeatedly. Kinetics were somewhat modified in the coupled enzyme as compared to the enzyme in solution. Glutamate appeared more available while oxaloacetate seemed to be limiting. These modifications might be due to the proteic support itself. The enzymatic films also revealed themselves as a good tool for industrial or clinical purposes as well as for studying the mechanism of enzyme action.     

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.     

Synthesis and biological evaluation of novel flavonoid derivatives as dual binding acetylcholinesterase inhibitors

A new series of flavonoid derivatives have been designed, synthesised and evaluated as acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. Among them, fifteen derivatives were found to inhibit the enzyme in the micromolar range and isoflavone derivatives possessed more potent inhibitory activity than other flavonoid derivatives. The best compound 9a had its inhibitory activity (IC(50) = 0.093 microM) in the same range as the reference compound, donepezil (IC(50) = 0.025 microM). Preliminary structure-activity relationships and a molecular modeling study for 9a have revealed that the isoflavone moiety plays a key role in the interaction of this series of derivatives with AChE by acting as an anchor in its peripheral anionic site.     

Dipeptide synthesis catalyzed by aminoacyl-tRNA synthetases from Bacillus stearothermophilus

A new approach to enzymatic peptide synthesis by using aminoacyl-tRNA synthetase (ARS) as a catalyst has been investigated. Four ARSs (AspRS, HisRS, LeuRS and TyrRS) have been purified from a thermophilic bacterium, Bacillus stearothermophilus. By using TyrRS as a catalyst, tyrosine and leucinamide were shown to be condensed in the presence of ATP to give tyrosylleucinamide. In this manner, all of the ARSs investigated catalyzed the peptide synthesis reactions. TyrRS did not have strict specificity for the amino acid derivatives used as substrates and even D-amino acids were incorporated into peptides fairly easily in this enzymatic reaction. Preparative scale synthesis of L-Tyr-L-LeuNH2 was carried out and from this the scope and limitation of this new enzymatic reaction as a tool to the peptide synthesis has been described.     

Animal and cellular models of Gaucher's disease. I. Refractoriness of thio analogs of glucocerebroside to enzymatic hydrolysis

In order to develop a nonmetabolizable analog of glucocerebroside to investigate the distribution and accumulation of this lipid in model systems, thiohemiacetal derivatives were synthesized and their susceptibility to enzymatic hydrolysis by purified human placental glucocerebrosidase was examined. Sulfur analogs were found to be completely refractory to the activity of this enzyme, indicating their potential use in animal and isolated cell models and possibly for the preparation of affinity chromatography columns for the isolation of glucocerebrosidase.     

Enzymatic Synthesis of Polyol- and Masked Polyol- Glycosides using β-Glycosidase of Sulfolobus Solfataricus

The use of commercially available mesophilic glycosidases in the enzymatic synthesis of glycosides of different types is a well established method suffering from some drawbacks such as a poor yield. Substrates with three or four hydroxyl groups have been subjected to enzymatic glucosylation using crude homogenate of the thermophilic archaeon Sulfolobus solfataricus containing a β-glycosidase activity able to transfer glucose, galactose and fucose from different donors. The stereochemistry of this reaction was interpreted in terms of interaction with a possible “glucose” active site of the enzyme. In addition masked or protected derivatives of tetritols and some simple unsaturated alcohols were glycosylated yielding glycosides in yields very competitive with those obtained using mesophilic enzymes, examples of further chemical manipulation of these compounds were reported. When using a scarce amount of acceptor, a reasonable amount of products could be obtained by adding different aliquots of donor at time intervals.     

Characterization of the zinc binding site of bacterial phosphotriesterase.

The bacterial phosphotriesterase has been found to require a divalent cation for enzymatic activity. This enzyme catalyzes the detoxification of organophosphorus insecticides and nerve agents. In an Escherichia coli expression system significantly higher concentrations of active enzyme could be produced when 1.0 mM concentrations of Mn2+, Co2+, Ni2+, and Cd2+ were included in the growth medium. The isolated enzymes contained up to 2 equivalents of these metal ions as determined by atomic absorption spectroscopy. The catalytic activity of the various metal enzyme derivatives was lost upon incubation with EDTA, 1,10-phenanthroline, and 8-hydroxyquinoline-5-sulfonic acid. Protection against inactivation by metal chelation was afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site. Apoenzyme was prepared by incubation of the phosphotriesterase with beta-mercaptoethanol and EDTA for 2 days. Full recovery of enzymatic activity could be obtained by incubation of the apoenzyme with 2 equivalents of Zn2+, Co2+, Ni2+, Cd2+, or Mn2+. The 113Cd NMR spectrum of enzyme containing 2 equivalents of 113Cd2+ showed two resonances at 120 and 215 ppm downfield from Cd(ClO4)2. The NMR data are consistent with nitrogen (histidine) and oxygen ligands to the metal centers.     

Novel cofactor derivatives and cofactor-based models

In 1997 and the first half of 1998, numerous publications appeared reporting studies of cofactors and their analogues in classical model systems and in enzyme-catalyzed reactions directed at understanding the enzymatic reactions of their natural cofactors. Model systems based on flavins have provided new insights into enzymatic modulation of the flavin reduction potential, and enzymatic reactions of coenzyme A analogues and derivatives have been employed in several studies of coenzyme A utilizing enzymes. Coenzyme B₁₂ analogues have been utilized as alternate cofactors for B₁₂-utilizing enzymes, while pyrroloquinoline quinone esters and analogues have been employed in model studies of the reactions of quinoprotein-catalyzed reactions.     

Denitrification and nitrite reduction: Pseudomonas aeruginosa nitrite-reductase

Present knowledge of the different enzymatic steps of the denitrification chains in various bacteria, particularly Paracoccus denitrificans and Pseudomonas aeruginosa has been briefly reviewed. The question whether nitric oxide (NO), nitrous oxide (N2O) and other nitrogen derivatives are obligatory intermediates has been discussed. The second part is an extensive review of the structure and the function of a key enzyme in denitrification, cytochrome c551-nitrite-oxidoreductase from P. aeruginosa. Recent results on the stoichiometry of nitrite reduction have been discussed.     

Modeling cellulase kinetics on lignocellulosic substrates

The enzymatic hydrolysis of cellulose to glucose by cellulases is one of the major steps involved in the conversion of lignocellulosic biomass to yield biofuel. This hydrolysis by cellulases, a heterogeneous reaction, currently suffers from some major limitations, most importantly a dramatic rate slowdown at high degrees of conversion. To render the process economically viable, increases in hydrolysis rates and yields are necessary and require improvement both in enzymes (via protein engineering) and processing, i.e. optimization of reaction conditions, reactor design, enzyme and substrate cocktail compositions, enzyme recycling and recovery strategies. Advances in both areas in turn strongly depend on the progress in the accurate quantification of substrate–enzyme interactions and causes for the rate slowdown. The past five years have seen a significant increase in the number of studies on the kinetics of the enzymatic hydrolysis of cellulose. This review provides an overview of the models published thus far, classifies and tabulates these models, and presents an analysis of their basic assumptions. While the exact mechanism of cellulases on lignocellulosic biomass is not completely understood yet, models in the literature have elucidated various factors affecting the enzymatic rates and activities. Different assumptions regarding rate-limiting factors and basic substrate–enzyme interactions were employed to develop and validate these models. However, the models need to be further tested against additional experimental data to validate or disprove any underlying hypothesis. It should also provide better insight on additional parameters required in the case that more substrate and enzyme properties are to be included in a model.