Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 56
Filter
Add more filters










Publication year range
1.
Methods Enzymol ; 699: 1-23, 2024.
Article in English | MEDLINE | ID: mdl-38942500

ABSTRACT

Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.


Subject(s)
Alkyl and Aryl Transferases , Cryoelectron Microscopy , Penicillium , Penicillium/enzymology , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/metabolism , Alkyl and Aryl Transferases/isolation & purification , Cryoelectron Microscopy/methods , Diterpenes/metabolism , Diterpenes/chemistry , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Dimethylallyltranstransferase/metabolism , Dimethylallyltranstransferase/chemistry , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/isolation & purification
2.
Angew Chem Int Ed Engl ; 60(15): 8460-8465, 2021 04 06.
Article in English | MEDLINE | ID: mdl-33586286

ABSTRACT

Cyanobactins comprise a widespread group of peptide metabolites produced by cyanobacteria that are often diversified by post-translational prenylation. Several enzymes have been identified in cyanobactin biosynthetic pathways that carry out chemically diverse prenylation reactions, representing a resource for the discovery of post-translational alkylating agents. Here, genome mining was used to identify orphan cyanobactin prenyltransferases, leading to the isolation of tolypamide from the freshwater cyanobacterium Tolypothrix sp. The structure of tolypamide was confirmed by spectroscopic methods, degradation, and enzymatic total synthesis. Tolypamide is forward-prenylated on a threonine residue, representing an unprecedented post-translational modification. Biochemical characterization of the cognate enzyme TolF revealed a prenyltransferase with strict selectivity for forward O-prenylation of serine or threonine but with relaxed substrate selectivity for flanking peptide sequences. Since cyanobactin pathways often exhibit exceptionally broad substrate tolerance, these enzymes represent robust tools for synthetic biology.


Subject(s)
Bacterial Proteins/chemistry , Dimethylallyltranstransferase/chemistry , Peptides, Cyclic/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Cyanobacteria/enzymology , Dimethylallyltranstransferase/isolation & purification , Dimethylallyltranstransferase/metabolism , Molecular Structure , Peptides, Cyclic/metabolism , Threonine/chemistry , Threonine/metabolism
3.
Commun Biol ; 2: 384, 2019.
Article in English | MEDLINE | ID: mdl-31646187

ABSTRACT

Plants produce various prenylated phenolic metabolites, including flavonoids, phloroglucinols, and coumarins, many of which have multiple prenyl moieties and display various biological activities. Prenylated phenylpropanes, such as artepillin C (3,5-diprenyl-p-coumaric acid), exhibit a broad range of pharmaceutical effects. To date, however, no prenyltransferases (PTs) involved in the biosynthesis of phenylpropanes and no plant enzymes that introduce multiple prenyl residues to native substrates with different regio-specificities have been identified. This study describes the isolation from Artemisia capillaris of a phenylpropane-specific PT gene, AcPT1, belonging to UbiA superfamily. This gene encodes a membrane-bound enzyme, which accepts p-coumaric acid as its specific substrate and transfers two prenyl residues stepwise to yield artepillin C. These findings provide novel insights into the molecular evolution of this gene family, contributing to the chemical diversification of plant specialized metabolites. These results also enabled the design of a yeast platform for the synthetic biology of artepillin C.


Subject(s)
Artemisia/enzymology , Dimethylallyltranstransferase/isolation & purification , Phenylpropionates/metabolism , Plant Proteins/isolation & purification , Artemisia/genetics , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Genes, Plant , Phenylpropionates/chemistry , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Prenylation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Substrate Specificity , Synthetic Biology/methods
4.
J Am Chem Soc ; 140(51): 17840-17845, 2018 12 26.
Article in English | MEDLINE | ID: mdl-30525563

ABSTRACT

The biosynthetic route to the napyradiomycin family of bacterial meroterpenoids has been fully described 32 years following their original isolation and 11 years after their gene cluster discovery. The antimicrobial and cytotoxic natural products napyradiomycins A1 and B1 are produced using three organic substrates (1,3,6,8-tetrahydroxynaphthalene, dimethylallyl pyrophosphate, and geranyl pyrophosphate), and catalysis via five enzymes: two aromatic prenyltransferases (NapT8 and T9); and three vanadium dependent haloperoxidase (VHPO) homologues (NapH1, H3, and H4). Building upon the previous characterization of NapH1, H3, and T8, we herein describe the initial (NapT9, H1) and final (NapH4) steps required for napyradiomycin construction. This remarkably streamlined biosynthesis highlights the utility of VHPO enzymology in complex natural product generation, as NapH4 efficiently performs a unique chloronium-induced terpenoid cyclization to establish two stereocenters and a new carbon-carbon bond, and dual-acting NapH1 catalyzes chlorination and etherification reactions at two distinct stages of the pathway. Moreover, we employed recombinant napyradiomycin biosynthetic enzymes to chemoenzymatically synthesize milligram quantities in one pot in 1 day. This method represents a viable enantioselective approach to produce complex halogenated metabolites, like napyradiomycin B1, that have yet to be chemically synthesized.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/chemistry , Dimethylallyltranstransferase/chemistry , Peroxidases/chemistry , Bacterial Proteins/isolation & purification , Biocatalysis , Dimethylallyltranstransferase/isolation & purification , Naphthoquinones/chemical synthesis , Peroxidases/isolation & purification , Streptomyces/enzymology
5.
Sci Rep ; 6: 24819, 2016 Apr 21.
Article in English | MEDLINE | ID: mdl-27098599

ABSTRACT

Due to their impressive pharmaceutical activities and safety, prenylated flavonoids have a high potent to be applied as medicines and nutraceuticals. Biocatalysis is an effective technique to synthesize prenylated flavonoids. The major concern of this technique is that the microbe-derived prenyltransferases usually have poor regiospecificity and generate multiple prenylated products. In this work, a highly regiospecific prenyltransferase (FoPT1) was found from Fusarium oxysporum. It could recognize apigenin, naringenin, genistein, dihydrogenistein, kampferol, luteolin and hesperetin as substrates, and only 6-C-prenylated flavonoids were detected as the products. The catalytic efficiency of FoPT1 on flavonoids was in a decreasing order with hesperetin >naringenin >apigenin >genistein >luteolin >dihydrogenistein >kaempferol. Chalcones, flavanols and stilbenes were not active when acting as the substrates. 5,7-Dihydroxy and 4-carbonyl groups of flavonid were required for the catalysis. 2,3-Alkenyl was beneficial to the catalysis whereas 3-hydroxy impaired the prenylation reaction. Docking studies simulated the prenyl transfer reaction of FoPT1. E186 was involved in the formation of prenyl carbonium ion. E98, F89, F182, Y197 and E246 positioned apigenin for catalysis.


Subject(s)
Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Flavonoids/biosynthesis , Fusarium/genetics , Fusarium/metabolism , Amino Acid Sequence , Biocatalysis , Cloning, Molecular , Codon , Dimethylallyltranstransferase/isolation & purification , Enzyme Activation , Flavonoids/chemistry , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Metals , Models, Molecular , Phylogeny , Protein Conformation , Recombinant Proteins , Substrate Specificity
6.
Appl Microbiol Biotechnol ; 100(12): 5389-99, 2016 Jun.
Article in English | MEDLINE | ID: mdl-26875876

ABSTRACT

FgaPT2 from Aspergillus fumigatus catalyzes a Friedel-Crafts alkylation at C-4 of L-tryptophan and is involved in the biosynthesis of the ergot alkaloids fumigaclavines. Several tryptophan-containing cyclic dipeptides had also been prenylated by FgaPT2, but the turnover rate (k cat) was low. Here, we report the generation of FgaPT2 mutants by saturation mutagenesis at the amino acid residue Arg244 to improve its catalytic efficiency toward cyclic dipeptides. Thirteen mutated enzymes demonstrated up to 76-fold higher turnover number toward seven cyclic dipeptides than the non-mutated FgaPT2. More importantly, the mutated enzymes exhibited different preferences toward these substrates. This study provides a convenient approach for creation of new biocatalysts for production of C4-prenylated cyclic dipeptides.


Subject(s)
Aspergillus fumigatus/enzymology , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Dipeptides/metabolism , Mutagenesis , Tryptophan , Arginine/genetics , Aspergillus fumigatus/metabolism , Biocatalysis , Dimethylallyltranstransferase/isolation & purification , Ergot Alkaloids , Kinetics , Magnetic Resonance Spectroscopy , Prenylation , Substrate Specificity
7.
Appl Microbiol Biotechnol ; 98(1): 199-206, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23525886

ABSTRACT

Paxilline is an indole-diterpene produced by Penicillium paxilli. Six genes (paxB, C, G, M, P, and Q) in paxilline biosynthetic gene cluster were previously shown to be responsible for paxilline biosynthesis. In this study, we have characterized paxD, which is located next to paxQ and has weak similarities to fungal dimethylallyl tryptophan synthase genes. PaxD was overexpressed in Escherichia coli and the purified enzyme was used for in vitro analysis. When paxilline and dimethylallyl diphosphate were used as substrates, one major and one minor product, which were identified as di-prenyl paxilline and mono-prenyl paxilline by liquid chromatography-mass spectrometry analysis, were formed. The structure of the major product was determined to be 21,22-diprenylated paxilline, showing that PaxD catalyzed the successive di-prenylation. Traces of both products were detected in culture broth of P. paxilli by liquid chromatography-mass spectrometry analysis. The enzyme is likely to be a dimer and required no divalent cations. The optimum pH and temperature were 8.0 and 37 °C, respectively. The Km values were calculated as 106.4 ± 5.4 µM for paxilline and 0.57 ± 0.02 µM for DMAPP with a kcat of 0.97 ± 0.01/s.


Subject(s)
Biosynthetic Pathways , Dimethylallyltranstransferase/metabolism , Indoles/metabolism , Multigene Family , Penicillium/enzymology , Chromatography, Liquid , Cloning, Molecular , Dimethylallyltranstransferase/chemistry , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/isolation & purification , Enzyme Stability , Escherichia coli/genetics , Gene Expression , Hemiterpenes/metabolism , Hydrogen-Ion Concentration , Mass Spectrometry , Organophosphorus Compounds/metabolism , Penicillium/genetics , Penicillium/metabolism , Prenylation , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Temperature
8.
Org Lett ; 15(22): 5834-7, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24188078

ABSTRACT

A big challenge in organic synthesis is to reach a high regioselectivity. Enzymes catalyze usually highly regiospecific reactions and can function as ideal biocatalysts for such purposes. Some secondary metabolite enzymes can even use distinctly different unnatural substrates and expand therefore their potential usage in chemoenzymatic synthesis. We report here the acceptance of benzyl diphosphate as an alkyl donor by the fungal dimethylallyl transferase FgaPT2 and the regiospecific enzymatic benzylation of tryptophan and several analogues.


Subject(s)
Aspergillus fumigatus/chemistry , Benzyl Compounds/chemistry , Dimethylallyltranstransferase/chemistry , Tryptophan/chemistry , Aspergillus fumigatus/enzymology , Catalysis , Dimethylallyltranstransferase/isolation & purification , Dimethylallyltranstransferase/metabolism , Molecular Structure , Tryptophan/metabolism
9.
Microbiology (Reading) ; 159(Pt 10): 2169-2179, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23845975

ABSTRACT

A putative prenyltransferase gene, NFIA_043650, was amplified from Neosartorya fischeri NRRL 181 and cloned into the expression vector pQE60. The deduced polypeptide consisting of 445 amino acids with a molecular mass of 51 kDa was overproduced in Escherichia coli and purified as His6-tagged protein to near homogeneity. The purified soluble protein was subsequently assayed with potential aromatic substrates in the presence of dimethylallyl diphosphate. HPLC analysis of the reaction mixtures revealed acceptance of all tested tryptophan-containing cyclic dipeptides. Isolation and structural elucidation of enzyme products of five selected substrates indicated a reverse C2-prenylation on the indole nucleus, proving the enzyme to be a cyclic dipeptide C2-prenyltransferase (CdpC2PT). Differing significantly from two known brevianamide F reverse C2-prenyltransferases NotF and BrePT which use cyclo-l-Trp-l-Pro as their preferred substrate, CdpC2PT showed a clear substrate preference for (S)-benzodiazepinedinone and cyclo-l-Trp-l-Trp with KM values of 84.1 and 165.2 µM and turnover numbers at 0.63 and 0.30 s(-1), respectively. A possible role of CdpC2PT in the biosynthesis of fellutanines is discussed.


Subject(s)
Dimethylallyltranstransferase/isolation & purification , Dimethylallyltranstransferase/metabolism , Neosartorya/enzymology , Chromatography, High Pressure Liquid , Cloning, Molecular , Dimethylallyltranstransferase/chemistry , Dimethylallyltranstransferase/genetics , Escherichia coli/genetics , Gene Expression , Kinetics , Molecular Sequence Data , Molecular Weight , Neosartorya/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Substrate Specificity
10.
FEBS J ; 280(11): 2572-80, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23490165

ABSTRACT

Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 µg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.


Subject(s)
Alkyl and Aryl Transferases/isolation & purification , Alkyl and Aryl Transferases/metabolism , Dimethylallyltranstransferase/isolation & purification , Dimethylallyltranstransferase/metabolism , Lithospermum/enzymology , Animals , Dimethylallyltranstransferase/genetics , Kinetics , Lithospermum/genetics , Plant Proteins/genetics , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Point Mutation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sf9 Cells , Spodoptera , Substrate Specificity
11.
Org Biomol Chem ; 10(46): 9262-70, 2012 Dec 14.
Article in English | MEDLINE | ID: mdl-23090579

ABSTRACT

The prenyltransferase FtmPT1 from Aspergillus fumigatus is involved in the biosynthesis of fumitremorgin-type alkaloids and catalysed the regular C2-prenylation of brevianamide F (cyclo-L-Trp-L-Pro). It has been shown that FtmPT1 also accepted a number of other tryptophan-containing cyclic dipeptides and prenylated them, in the presence of dimethylallyl diphosphate, at C-2 of the indole nucleus. Detailed analysis of the incubation mixtures of FtmPT1 with these cyclic dipeptides revealed the presence of additional product peaks in the HPLC chromatograms. Seven regularly C3-prenylated hexahydropyrrolo[2,3-b]indoles were isolated and identified by HR-ESI-MS and NMR analyses including HMBC, HMQC and NOESY experiments. Further experiments proved that the C2- and C3-prenylated products are both independent enzyme products. To the best of our knowledge, this is the first report on the enzymatic formation of regularly C3-prenylated indolines. A reaction mechanism for both C2- and C3-prenylated derivatives was proposed.


Subject(s)
Aspergillus fumigatus/enzymology , Dimethylallyltranstransferase/chemistry , Hemiterpenes/chemistry , Indoles/chemistry , Organophosphorus Compounds/chemistry , Peptides, Cyclic/chemistry , Aspergillus fumigatus/chemistry , Chromatography, High Pressure Liquid , Dimethylallyltranstransferase/isolation & purification , Escherichia coli/genetics , Magnetic Resonance Spectroscopy , Prenylation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Spectrometry, Mass, Electrospray Ionization , Stereoisomerism , Substrate Specificity
12.
Biosci Biotechnol Biochem ; 76(7): 1389-93, 2012.
Article in English | MEDLINE | ID: mdl-22785469

ABSTRACT

Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.


Subject(s)
Citrus/enzymology , Coumarins/metabolism , Dimethylallyltranstransferase/isolation & purification , Furocoumarins/metabolism , Geranyltranstransferase/isolation & purification , Plant Proteins/isolation & purification , Cations, Divalent/chemistry , Cations, Divalent/metabolism , Chromatography, High Pressure Liquid , Citrus/chemistry , Coumarins/chemistry , Dimethylallyltranstransferase/metabolism , Diphosphates/chemistry , Diphosphates/metabolism , Diterpenes/chemistry , Diterpenes/metabolism , Furocoumarins/chemistry , Geranyltranstransferase/metabolism , Humans , Kinetics , Microsomes/chemistry , Microsomes/enzymology , Plant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity
13.
Article in English | MEDLINE | ID: mdl-22442243

ABSTRACT

CdpNPT from Aspergillus fumigatus is a dimethylallyltryptophan synthase/indole prenyltransferase that catalyzes reverse prenylation at position N1 of tryptophan-containing cyclic dipeptides. Residues 38-440 of CdpNPT were expressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion and microseeding techniques. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.4, b = 157.1, c = 161.8 Å, α = ß = γ = 90.0°.


Subject(s)
Aspergillus fumigatus/enzymology , Dimethylallyltranstransferase/chemistry , Crystallization , Crystallography, X-Ray , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/isolation & purification , Gene Expression
14.
J Biol Chem ; 287(2): 1371-80, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22123822

ABSTRACT

The putative prenyltransferase gene ACLA_031240 belonging to the dimethylallyltryptophan synthase superfamily was identified in the genome sequence of Aspergillus clavatus and overexpressed in Escherichia coli. The soluble His-tagged protein EAW08391 was purified to near homogeneity and used for biochemical investigation with diverse aromatic substrates in the presence of different prenyl diphosphates. It has shown that in the presence of dimethylallyl diphosphate (DMAPP), the recombinant enzyme accepted very well simple indole derivatives with L-tryptophan as the best substrate. Product formation was also observed for tryptophan-containing cyclic dipeptides but with much lower conversion yields. In contrast, no product formation was detected in the reaction mixtures of L-tryptophan with geranyl or farnesyl diphosphate. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific regular prenylation at C-5 of the indole nucleus of the simple indole derivatives. EAW08391 was therefore termed 5-dimethylallyltryptophan synthase, and it filled the last gap in the toolbox of indole prenyltransferases regarding their prenylation positions. K(m) values of 5-dimethylallyltryptophan synthase were determined for L-tryptophan and DMAPP at 34 and 76 µM, respectively. Average turnover number (k(cat)) at 1.1 s(-1) was calculated from kinetic data of L-tryptophan and DMAPP. Catalytic efficiencies of 5-dimethylallyltryptophan synthase for L-tryptophan at 25,588 s(-1)·M(-1) and for other 11 simple indole derivatives up to 1538 s(-1)·M(-1) provided evidence for its potential usage as a catalyst for chemoenzymatic synthesis.


Subject(s)
Aspergillus/enzymology , Dimethylallyltranstransferase/chemistry , Fungal Proteins/chemistry , Hemiterpenes/chemistry , Organophosphorus Compounds/chemistry , Aspergillus/genetics , Catalysis , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/isolation & purification , Dimethylallyltranstransferase/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Hemiterpenes/metabolism , Organophosphorus Compounds/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity/physiology
15.
Bioorg Med Chem ; 17(16): 6003-7, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19615911

ABSTRACT

Aromatic substrates tyrosol (p-hydroxyphenylethanol) and 2,6-dihydroxynaphthalene (2,6-DHN) were converted into chromane derivatives by means of chemoenzymatic reactions catalyzed by the aromatic prenyltransferase of bacterial origin NovQ, using dimethylallyl bromide as allylic substrate instead of the natural isoprenyl pyrophosphate substrate. Stereoselective prenylation occurred in o-position with respect to the phenol hydroxyl in both compounds. Prenylated derivatives were readily converted into chromane products via a selective 6-endo-trig cyclization involving the oxygen atom from the phenol moiety and the double bond of the prenyl substituent, a process catalyzed by FeCl(3). These findings set up the basis of a most convenient two-step, one-pot process which allows for easy recovery of the chromane products in high yields. The chromane derivatives thus obtained were tested for cytotoxicity and pro-apoptotic activity using LoVo WT cells, a line of human colon adenocarcinoma.


Subject(s)
Antineoplastic Agents/chemistry , Chromans/chemistry , Dimethylallyltranstransferase/metabolism , Growth Inhibitors/biosynthesis , Antineoplastic Agents/metabolism , Antineoplastic Agents/toxicity , Biocatalysis , Cell Line, Tumor , Chromans/toxicity , Colonic Neoplasms/drug therapy , Dimethylallyltranstransferase/chemistry , Dimethylallyltranstransferase/isolation & purification , Growth Inhibitors/chemistry , Growth Inhibitors/toxicity , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
16.
J Biol Chem ; 284(21): 14439-47, 2009 May 22.
Article in English | MEDLINE | ID: mdl-19339241

ABSTRACT

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C-C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The K(m) value for dimethylallyl diphosphate was determined as 116 microm. For dihydro-PCA, half-maximal velocity was observed at 35 microm. K(cat) was calculated as 0.435 s(-1). PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.


Subject(s)
Bacterial Proteins/metabolism , Dimethylallyltranstransferase/metabolism , Phenazines/metabolism , Prenylation , Streptomyces/enzymology , Bacterial Proteins/isolation & purification , Carboxylic Acids/chemistry , Chromatography, High Pressure Liquid , Cloning, Molecular , Dimethylallyltranstransferase/isolation & purification , Gene Silencing , Genes, Bacterial , Molecular Sequence Data , Multigene Family , Phenanthrenes/chemistry , Phenazines/chemistry , Streptomyces/genetics , Streptomyces coelicolor/metabolism
17.
FEBS Lett ; 581(16): 2889-93, 2007 Jun 26.
Article in English | MEDLINE | ID: mdl-17543953

ABSTRACT

Fnq26 from Streptomyces cinnamonensis DSM 1042 is a new member of the recently identified CloQ/Orf2 class of prenyltransferases. The enzyme was overexpressed in E. coli and purified to apparent homogeneity, resulting in a soluble, monomeric protein of 33.2 kDa. The catalytic activity of Fnq26 is independent of the presence of Mg(2+) or other divalent metal ions. With flaviolin (2,5,7-trihydroxy-1,4-naphthoquinone) as substrate, Fnq26 catalyzes the formation of a carbon-carbon-bond between C-3 (rather than C-1) of geranyl diphosphate and C-3 of flaviolin, i.e. an unusual "reverse" prenylation. With 1,3-dihydroxynaphthalene and 4-hydroxybenzoate as substrates Fnq26 catalyzes O-prenylations.


Subject(s)
Amino Acids, Aromatic/metabolism , Dimethylallyltranstransferase/metabolism , Magnesium/pharmacology , Protein Prenylation , Streptomyces/enzymology , Catalysis , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/isolation & purification , Escherichia coli/genetics , Models, Biological , Protein Prenylation/drug effects , Sequence Analysis, Protein , Solubility , Substrate Specificity , Transformation, Bacterial
18.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 62(Pt 11): 1153-5, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-17077503

ABSTRACT

Crystals of recombinant CloQ (subunit MW = 35 626 Da; 324 amino acids), an aromatic prenyltransferase from Streptomyces roseochromogenes, were grown by vapour diffusion. The protein crystallizes in space group I4(1)22, with unit-cell parameters a = b = 135.19, c = 98.13 A. Native data from a single crystal were recorded to a resolution of 2.2 A in-house. Preliminary analysis of these data indicated that the asymmetric unit corresponds to a monomer, giving an estimated solvent content of 60.6%. CloQ is involved in the biosynthesis of the aminocoumarin antibiotic clorobiocin, which targets the essential bacterial enzyme DNA gyrase.


Subject(s)
Dimethylallyltranstransferase/chemistry , Novobiocin/analogs & derivatives , Streptomyces/enzymology , Anti-Bacterial Agents/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Crystallography, X-Ray , Dimethylallyltranstransferase/isolation & purification , Novobiocin/biosynthesis
19.
Chembiochem ; 7(1): 158-64, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16397874

ABSTRACT

A putative prenyltransferase gene-fgaPT1-has been identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. The gene was cloned and overexpressed in Escherichia coli, and the His6-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A into fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, that is, by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined to be 6 and 13 microM, respectively, while the turnover number was 0.8 s(-1). Metal ions such as Mg2+ and Ca2+ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A, with only dimethylallyl diphosphate being accepted as a donor under our conditions. FgaPT1 is the first reverse prenyltransferase from fungi to have been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus.


Subject(s)
Aspergillus fumigatus/chemistry , Aspergillus fumigatus/enzymology , Dimethylallyltranstransferase , Gene Expression Regulation, Enzymologic , Indole Alkaloids/metabolism , Aspergillus fumigatus/metabolism , Claviceps/chemistry , Claviceps/metabolism , Cloning, Molecular , Dimethylallyltranstransferase/chemistry , Dimethylallyltranstransferase/isolation & purification , Dimethylallyltranstransferase/metabolism , Ergot Alkaloids , Indole Alkaloids/chemistry , Molecular Structure , Sequence Analysis, DNA , Substrate Specificity , Time Factors
20.
Trends Pharmacol Sci ; 26(12): 606-8, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16229901

ABSTRACT

The recent identification of genes encoding a novel family of soluble aromatic prenyltransferases from Streptomyces species and the structural characterization of one of these proteins are challenging discoveries in the biochemistry of natural compounds. In addition to their notable physiological role in the biosynthesis of aminocoumarin antibiotics, these enzymes represent an advanced tool for exploring novel isoprenoid substitutions in aromatic compounds and are expected to foster the emerging pharmacological applications of prenylated flavonoids.


Subject(s)
Dimethylallyltranstransferase/chemistry , Flavonoids/chemistry , Protein Prenylation , Streptomyces/enzymology , Dimethylallyltranstransferase/isolation & purification , Substrate Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...