Plant-Derived Enzymes Producing Chiral Aroma Compounds and Potential Application

Aroma (volatile) compounds play important ecological functions in plants, and also contribute to the quality of plant-derived foods. Moreover, chiral aroma compounds affect their functions in plants and lead to different flavor quality properties. Formations of chiral aroma compounds are due to the presence of enzymes producing these compounds in plants, which are generally involved in the final biosynthetic step of the aroma compounds. Here, we review recent progress in research on the plant-derived enzymes producing chiral aroma compounds, and their changes in response to environmental factors. The chiral aroma enzymes that have been reported produce (R)-linalool, (S)-linalool, (R)-limonene, and (S)-limonene, etc., and these enzymes are found in various plant species. We also discuss the origins of enantioselectivity in the plant-derived enzymes producing chiral aroma compounds and summarize the potential use of plants containing enzymes producing chiral aroma compounds for producing chiral flavors/fragrances.     

Hydroxylated serotonin and dopamine as substrates and inhibitors for human cytosolic SULT1A3

Sulfation as catalyzed by the cytosolic sulfotransferases (SULTs) is known to play an important role in the regulation and homeostasis of monoamine neurotransmitters. The current study was designed to examine the occurrence of the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine by human cytosolic SULTs and to investigate the inhibitory effects of these hydroxylated derivatives on the sulfation of their unhydroxylated counterparts, serotonin and dopamine. A systematic study using 11 known human cytosolic SULTs revealed SULT1A3 as the responsible enzyme for the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine. The pH-dependence and kinetic constants of SULT1A3 with 7-hydroxyserotonin or 6-hydroxydopamine as substrate were determined. The inhibitory effects of 7-hydroxyserotonin and 6-hydroxydopamine on the sulfation of serotonin and dopamine were evaluated. Kinetic analyses indicated that the mechanism underlying the inhibition by these hydroxylated monoamine derivatives is of a competitive-type. Metabolic labeling experiments showed the generation and release of [³⁵S]sulfated 7-hydroxyserotonin and [³⁵S]sulfated 6-hydroxydopamine when SK-N-MC human neuroblastoma cells were labeled with [³⁵S]sulfate in the presence of 7-hydroxyserotonin or 6-hydroxydopamine. Upon transfection of the cells with siRNAs targeted at SULT1A3, diminishment of the SULT1A3 protein and concomitantly the sulfating activity toward these hydroxylated monoamines was observed. Taken together, these results indicated clearly the involvement of sulfation in the metabolism of 7-hydroxyserotonin and 6-hydroxydopamine. By serving as substrates for SULT1A3, these hydroxylated monoamines may interfere with the homeostasis of endogenous serotonin and dopamine.     

Bicyclo((aryl)methyl)benzamides as inhibitors of GlyT1

A series of isoquinuclidine benzamides as glycine uptake inhibitors for the treatment of schizophrenia are described. Potency, lipophilicity, and intrinsic human microsomal clearance were parameters for optimization. Potency correlated with the nature of the ortho substituents of the benzamide ring, and reductions in lipophilicity could be achieved through heteroatom incorporation in the benzamide and pendant phenyl moieties. Improvements in human CL_int were achieved through changes in ring size and the N-alkyl group of the isoquinuclidine itself, with des-alkyl derivatives (40–41, 44) demonstrating the most robust microsomal stability. Dimethylbenzamide 9 was tested in a mouse MK801 LMA assay and had a statistically significant attenuation of locomotor activity at 3 and 10?μmol/kg compared to control.     

Binding conformations and QSAR of CA-4 analogs as tubulin inhibitors

A theoretical study on the binding conformations and the quantitative structure–activity relationship (QSAR) of combretastatin A4 (CA-4) analogs as inhibitors toward tubulin has been carried out using docking analysis and comparative molecular field analysis (CoMFA). The appropriate binding orientations and conformations of these compounds interacting with tubulin were revealed by the docking study; and a 3D-QSAR model showing significant statistical quality and satisfactory predictive ability was established, in which the correlation coefficient (R²) and cross-validation coefficient (q²) were 0.955 and 0.66, respectively. The same model was further applied to predict the pIC₅₀ values for 16 congeneric compounds as external test set, and the predictive correlation coefficient R²_pred reached 0.883. Other tests on additional validations further confirmed the satisfactory predictive power of the model. In this work, it was very interesting to find that the 3D topology structure of the active site of tubulin from the docking analysis was in good agreement with the 3D-QSAR model from CoMFA for this series of compounds. Some key structural factors of the compounds responsible for cytotoxicity were reasonably presented. These theoretical results can offer useful references for understanding the action mechanism and directing the molecular design of this kind of inhibitor with improved activity.     

Thiorphan, tiopronin, and related analogs as substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase (PAM)

Peptidyglycine alpha-amidating monooxygenase is a copper- and zinc-dependent, bifunctional enzyme that catalyzes the cleavage of glycine-extended peptides or N-acylglycines to the corresponding amides and glyoxylate. This reaction is a key step in the biosynthesis of bioactive alpha-amidated peptides and, perhaps, the primary fatty acids amides also. Two clinically useful N-acylglycines are thiorphan and tiopronin, each with a thiol moiety attached to the acyl group. We report here that thiorphan and tiopronin are substrates for PAM, exhibiting relatively low K(M,app) and V(MAX,app) values. The low V(MAX,app) values result, most likely, from a decrease in active PAM.2Cu(II) as the enzyme competes ineffectively with thiorphan and tiopronin for free copper.     

Geranyl and neryl triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase

When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase.     

Detergents as intrinsic P-glycoprotein substrates and inhibitors

We assessed the interaction of three electrically neutral detergents (Triton X-100, C₁₂EO₈, and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG_tl⁰, that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent-transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG_EO⁰ = − 1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.     

Oral contraceptives as substrates and inhibitors for human cytosolic SULTs

Cytosolic sulfotransferases (SULTs) in mammals are involved in the biotransformation and homeostasis of various endogenous and xenobiotic compounds. The current study aimed to examine the sulfation of contraceptive compounds by various human cytosolic SULTs and to investigate the inhibitory effects and mode of action of these compounds on the sulfation of 17beta-estradiol, a major endogenous estrogen. A systematic study using all eleven known human cytosolic SULTs revealed the differential substrate specificity of these enzymes for the eight representative contraceptive compounds and two endogenous estrogens (estrone and 17beta-estradiol) tested as substrates. Activity data showed that SULT1A1 displayed the strongest activity toward 17alpha-ethynylestradiol. Kinetic studies revealed that the V (max) value of the sulfation of 17alpha-ethynylestradiol by SULT1A1 was 1.64 times that of the sulfation of 17beta-estradiol, while the K (m) values were almost equal for the two compounds. The inhibitory effects of three contraceptive compounds on the sulfation of 17beta-estradiol by SULT1A1 were examined. IC(50) values determined were 0.193, 1.84, and 2.98 mM, respectively, for 19-norethindrone acetate, ethynodiol diacetate and mifepristone. Kinetic analyses indicated that the mechanism underlying the inhibition by these contraceptives is of a mixed noncompetitive type. Metabolic labeling experiments confirmed the sulfation of contraceptive compounds and the release of their sulfated derivatives by HepG2 human hepatoma cells. Collectively, the results obtained suggest a role of sulfation in the metabolism of contraceptive compounds in vivo. Moreover, in view of their inhibitory effects on the sulfation of 17beta-estradiol, these compounds may potentially act to disrupt the homeostasis of endogenous estrogens.     

Secondary alkyl hydroperoxides as inhibitors and alternate substrates for lipoxygenase

Lipoxygenase plays a central role in polyunsaturated fatty acid metabolism, inaugurating the biosynthesis of eicosanoids in animals and phytooxylipins in plants. Redox cycling of the non-heme iron cofactor represents a critical element of the catalytic mechanism. Paradoxically, the isolated enzyme contains Fe(II), but the catalytically active form contains Fe(III), and the natural oxidant for the iron is the hydroperoxide product of the catalyzed reaction. Controlling the redox state of lipoxygenase iron with small molecules, inhibitors or activators, could be a means to modulate the activity of the enzyme. The effects of secondary alkyl hydroperoxides and the corresponding alcohols on soybean lipoxygenase-1 reaction rates were investigated and found to be very different. Secondary alcohols were noncompetitive or linear mixed inhibitors with inhibition constants in the millimolar concentration range, with more hydrophobic compounds producing lower values. Secondary alkyl hydroperoxides were inhibitors of lipoxygenase-1 primarily at high substrate concentration. They were more effective inhibitors than the alcohols, with dissociation constants in the micromolar concentration range. The hydroperoxides bearing longer alkyl substituents were the more effective inhibitors. Oxidation of the iron in lipoxygenase-1 by 2-hydroperoxyalkanes was evident in electron paramagnetic resonance (EPR) measurements, but the enzyme was neither activated nor was it inactivated. Instead there was evidence for an entirely different reaction catalyzed by the enzyme, a homolytic dehydration of the hydroperoxide to produce the corresponding carbonyl compound.     

Enantioselectivity of inhibition of cytochrome P450 3A4 (CYP3A4) by ketoconazole: Testosterone and methadone as substrates

Racemic ketoconazole (KTZ) was the first orally active azole antifungal agent used in clinical practice and has become widely used in the treatment of mucosal fungal infections associated with AIDS immunosuppression and cancer chemotherapy. However, the use of KTZ has been limited because of adverse drug-drug interactions. KTZ blocks ergosterol biosynthesis by inhibiting the fungal cytochrome P450 (CYP51). KTZ is also a potent inhibitor of human cytochrome P450 3A4 (CYP3A4) enzyme, the major drug-metabolizing CYP isozyme in the human liver. We examined the enantioselective differences of KTZ in the inhibition of human CYP3A4 and in antifungal action. Dextro- and levo-KTZ exhibited modest enantioselective differences with respect to CYP3A4 inhibition of testosterone and methadone metabolism. For both substrates levo-KTZ was approximately a 2-fold more potent inhibitor. We examined the enantioselective differences in the in vitro activity of KTZ against medically relevant species of Candida and Aspergillus, as well as Cryptococcus neoformans. Overall, levo-KTZ was 2-4-fold more active than dextro-KTZ. Therefore, levo-KTZ is a more potent inhibitor of CYP3A4 and has stronger in vitro antifungal activity. Chirality 16:79-85, 2004.     

Hydroxamates as substrates and inhibitors for FMN-dependent 2-hydroxy acid dehydrogenases

Long-chain hydroxy acid oxydase (HAO) is a member of a flavoenzyme family with significant amino acid sequence similarity and strongly conserved three-dimensional structure; in particular, active-site amino acids involved in catalysis are invariant, with one exception, and numerous enzymatic studies suggest an identical chemical mechanism involving an intermediate carbanion for all family members. Known physiological substrates are a variety of L-2-hydroxy acids. Peroxisomal HAO differs from the other family members in that its actual physiological substrate is not known; it was first described as an L-amino acid oxidase, and recently was identified as an enzyme that converts creatol (hydroxycreatinine) to methylguanidine (a metabolite involved in a variety of uremic syndromes). Creatol (2-amino-5-hydroxy-1-methyl-4(5H)imidazolone) is not a 2-hydroxy acid. We show in this work that 2-hydroxyphenyl acetohydroxamate (HYPAH, the hydroxamate of mandelic acid), a compound that bears similarity both to mandelate (one of the best substrates known) and to creatol, is turned over by HAO, but between 10- and 100-fold less efficiently than mandelate itself. The compound also binds to the active site of homologous flavocytochrome b(2) (L-lactate dehydrogenase). Comparative pH-rate studies for mandelate and its hydroxamate suggest that HYPAH may bind in its ionized form. Both pH-rate profiles are bell-shaped curves, as are those determined for two other family members, flavocytochrome b(2) and mandelate dehydrogenase; while the group with an acid pK(a) between 5 and 6 is most likely the active-site histidine (the residue which abstracts the substrate C2 proton), the identity of the basic group is less clear. It has been proposed to be one of the active site arginines (Lehoux, I., and Mitra, B. (1999) Biochemistry38, 5836-5848); we suggest as an alternative that it could be the lysine residue that interacts with the flavin N1 and O2 positions and stabilizes the negative charge of reduced flavin. In addition to these studies, we have found that HAO is competitively inhibited by benzohydroxamate, which is one atom shorter than HYPAH; its affinity is nearly 100-fold lower than that of the substrate, in contrast to the strong inhibition it exerts on mandelate racemase (Maurice, St. M., and Bearne, S. L. (2000) Biochemistry39, 13324-13335). In the latter case, the 100-fold higher affinity compared to mandelate was proposed to arise from the fact that the hydroxamate can mimic the enolic intermediate which lies on the reaction pathway after C2 proton abstraction. Thus our results do not support the existence of a similar enolic intermediate for HAO (and probably its homologues), although they do not disprove it.     

Primary alcohols and di-alcohols as growth substrates for the purple nonsulfur bacterium Rhodobacter capsulatus

Growth experiments were performed with the purple nonsulfur bacterium Rhodobacter capsulatus to test its ability to use aliphatic, methyl-substituted, and unsaturated alcohols, as well as di-alcohols, as carbon sources for growth. Both phototrophic and chemotrophic growth was observed on a wide variety of such alcohols. By contrast, secondary or tertiary alcohols, or primary alcohols containing an ethyl or propyl substituent, did not support growth. In addition, preculture history and serial subculturing were found to be important factors for obtaining reliable growth of R. capsulatus on alcohols. Collectively, these results suggest that the carbon nutritional diversity of Rhodobacter capsulatus is even greater than previously suspected and that besides metabolizing organic acids and fatty acids in nature, this species may also be a major consumer of alcohols.     

Determination of amphetamine and methamphetamine enantiomers by chiral derivatization and gas chromatography–mass spectrometry as a test case for an automated sample preparation system

An automated sample preparation system has been applied to the chiral analysis of amphetamine and methamphetamine using derivatization with trifluoracetyl-L -prolyl chloride (L -TPC) and subsequent separation on a gas chromatography–mass spectrometry (GC-MS) system. Tasks automated were the dilution of standards and the off-line preparation of the diastereoisomer derivatives. Chromatographic performance, sensitivity, and reproducibility of the automated procedure were compared to the equivalent values obtained with two existing assays methods which employ manual derivatiation, either on-column or off-line. Chromatographic performance was unaffected by the derivatization procedure and sensitivity was better for both automated and manual off-line derivatization. Qualitative reproducibility as based on enantiomeric composition was equivalent for all three approaches, while quantitative reproducibility as based on peak areas was best for the automated procedure. Considering the fact that the diastereoisomer derivatives are unstable over time, automated sample preparation with “just-in-time” derivatization can increase the overall precision of the analytical method. The procedures described here are general enough in nature that they could be applied to other chiral or even achiral analytes. © 1994 Wiley-Liss, Inc.     

New chiral stationary phases for liquid chromatography based on small molecules: Development,enantioresolution evaluation and chiral recognition mechanisms

Recently, we reported the development of new chiral stationary phases (CSPs) for liquid chromatography (LC) based on chiral derivatives of xanthones (CDXs). Based on the most promising CDX selectors, 12 new CSPs were successfully prepared starting from suitable functionalized small molecules including xanthone and benzophenone derivatives. The chiral selectors comprising one, two, three, or four chiral moieties were covalently bonded to a chromatographic support and further packed into LC stainless-steel columns (150 × 2.1 mm I.D.). The enantioselective performance of the new CSPs was evaluated by LC using different classes of chiral compounds. Specificity for enantioseparation of some CDXs was observed in the evaluation of the new CSPs. Besides, assessment of chiral recognition mechanisms was performed by computational studies using molecular docking approach, which are in accordance with the chromatographic parameters. X-Ray analysis was used to establish a chiral selector 3D structure.     

Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase

Human estrogen sulfotransferase (SULT1E1) is involved in the regulation of 17beta-estradiol responsiveness and is believed to protect peripheral tissues from excessive estrogenic effects. Several assays already have been developed to investigate the inhibitory effect of endocrine-disrupting compounds (EDCs) on SULT1E1. However, most of these assays make use of the radiolabeled cofactor [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) or radiolabeled substrate [(3)H]estradiol. In this article, we describe the development and validation of an assay for the inhibition of human SULT1E1 that is rapid and simple and that uses the nonradioactive and noncarcinogenic 1-hydroxypyrene. A gradient HPLC separation of 15 min using a C18-RP column was developed to detect 1-hydroxypyrene and its metabolite pyrene 1-sulfate fluorescently. Time- and protein-dependent formation of pyrene 1-sulfate was investigated, and enzyme kinetics was determined (K(m)=6.4+/-0.8 nM and V(max)=158+/-19 pmol/min/microg SULT1E1). At higher 1-hydroxypyrene concentrations, the assay displayed non-Michaelis-Menten kinetics involving substrate inhibition. IC(50) values have been determined for eight known SULT1E1 inhibitors or competing substrates (17beta-estradiol, 17alpha-estradiol, genistein, 17alpha-ethynylestradiol, estrone, diethylstilbestrol, estriol, and hexestrol) and two previously unknown SULT1E1 inhibitors (zearalenone and dienestrol). The method was demonstrated to be easy, feasible, and highly reproducible for SULT1E1 screening assay inhibition studies.     

Williopsis californica, Williopsis saturnus, and Pachysolen tannophilus: novel microorganisms for stereoselective oxidation of secondary alcohols

A screening of 416 microorganisms from different taxonomical groups (bacteria, actinomycetes, yeasts, and filamentous fungi) has been performed looking for active strains in the stereoselective oxidation of secondary alcohols. The working collection was composed of 71 bacterial strains, 45 actinomycetes, 59 yeasts, 60 basidiomycetes, 33 marine fungi, and 148 filamentous fungi. All microorganisms selected were mesophilic. Yeasts were the most active microbial group in the whole-cell-catalyzed oxidation. Williopsis californica, Williopsis saturnus, and Pachysolen tannophilus were the strains of greatest interest, both as growing cells and as resting cells. The oxidation of the alcohols takes place when cells are in the stationary growth phase (after 48 h of culture). These three strains are S-stereoselective for the oxidation of racemic secondary alkanols and show stereospecificity in the oxidation of menthol or neo-menthol, whereas iso-menthol is not oxidized. In the case of the 1-tetrahydronaphtol enantiomers, only the S-enantiomer is oxidized. The three strains were immobilized by entrapment using agarose and agar from algae of the Gracilaria genus. The agarose derivatives displayed significant improvement in the stereospecificity of the reactions.     

Application of PEG-400 as a green biodegradable polymeric medium for the catalyst-free synthesis of spiro-dihydropyridines and their use as acetyl and butyrylcholinesterase inhibitors

A simple, efficient and green approach for the synthesis of spiro-dihydropyridines derivatives by one-pot multi-component reaction of isatin or acenaphthoquinone derivatives (1 equiv) with malononitrile (1 equiv) and N,N′-substituted-2-nitroethene-1,1-diamines (1 equiv) in PEG-400 under catalyst-free conditions is described. This method provides several advantages such as environmental friendliness, short reaction time, and simple workup procedure for the synthesis of biologically important compounds. The ability of synthesized compounds in inhibition of acetyl and butyrylcholinesterase were investigated both in vitro and in silico. All compounds showed moderate to high level activity against both acetyl and butyrylcholinesterase. There was a good correlation between in vitro and in silico studies.     

Some coumarins and benzoxazinones as potent paraoxonase 1 inhibitors

In this study, we aimed to investigate the effect of some coumarin and benzoxazinone derivatives on the activity of human PON1. Human serum paraoxonase 1 was purified from fresh human serum blood by two-step procedures that are ammonium sulfate precipitation (60–80%) and then hydrophobic interaction chromatography (Sepharose 4B, L-tyrosine and 1-napthylamine). The enzyme was purified 232-fold with a final specific activity of 27.1?U/mg. In vitro effects of some previously synthesized ionic coumarin or benzoxazinone derivatives (1–21) on purified PON1 activity were investigated. Compound 14 (1-(2,3,4,5,6)-pentamethylbenzyl-3-(6,8-dimethyl-2H-chromen-2-one-4-yl))benzimidazolium chloride was found out as the strongest inhibitor (IC₅₀?=?7.84?μM) for PON1 among the compounds. Kinetic investigation and molecular docking study were evaluated for one of the most active compounds (compound 12) and obtained data showed that this compound is competitive inhibitor of PON1 and interact with Leu262 and Ser263 in the active site of PON1. Moreover, coumarin derivatives were found out as the more potent inhibitors for PON1 than benzoxazinone derivatives.     

A colorimetric chiral sensor based on chiral crown ether for the recognition of the two enantiomers of primary amino alcohols and amines

A new colorimetric chiral sensor material consisting of three different functional sites such as chromophore (2,4-dinitrophenylazophenol dye), binding site (crown ether), and chiral barrier (3,3'-diphenyl-1,1'-binaphthyl group) was prepared and applied to the recognition of the two enantiomers of primary amino alcohols and amines. Among five primary amino alcohols and two primary amines tested, the two enantiomers of phenylalaninol show the highest difference in the absorption maximum wavelength (Δλ(max)=43.5 nm) and in the association constants (K(S)/K(R)=2.51) upon complexation with the colorimetric chiral sensor material and, consequently, the two enantiomers of phenylalaninol were clearly distinguished from each other by the color difference.