FgaPT2酶催化合成C-4异戊烯基化吲哚二酮哌嗪和定向诱变增强收率

在二甲基烯丙基二磷酸存在下,通过FgaPT2的酶催化合成C-4异戊烯化吲哚二酮哌嗪,对合成的产物进行了抗肿瘤、抗细菌、抗真菌、抗氧化活性测试,对生物活性最高的产物,研究了通过定点诱变提高酶合成产率的可行性。结果表明,FgaPT2酶催化合成了7个C4-异戊烯化吲哚二酮哌嗪,FgaPT2对底物具有一定的选择性,C4-异戊二烯化显著提高吲哚二酮哌嗪的生物活性,尤其是异戊二烯化产物 6b。Arg244的定点诱变表明,52.6%的FgaPT2突变体,提高了6b的合成产率,动力学参数验证了6a与突变FgaPT2之间的相互作用,可以提高异戊二烯基的合成产率。     

Chemoenzymatic synthesis of prenylated indole derivatives by using a 4-dimethylallyltryptophan synthase from Aspergillus fumigatus

A 4-dimethylallyltryptophan synthase, FgaPT2, has been identified in the genome of Aspergillus fumigatus. In a previous study, FgaPT2 was overexpressed in Saccharomyces cerevisiae and characterized biochemically. A higher protein yield (up to 100-fold higher than that for S. cerevisiae) has now been achieved by overexpression in E. coli; this has permitted investigation into substrate specificity with alternative substances. FgaPT2 accepted 17 of 37 commercially available indole derivatives as substrates. Tryptophan derivatives that carry methyl groups at the indole ring showed a different acceptance from those with methyl groups on the side chain. 5-Hydroxytryptophan was well accepted by FgaPT2, while the halogenated derivatives were not accepted. Decarboxylation, deamination, or oxidative deamination of tryptophan, as well as replacement of the NH(2) group by OH, or of the COOH group by CH(2)COOH or CONHOH resulted in decreased but still significant enzymatic activity. None of the tested tryptophan-containing dipeptides was accepted by FgaPT2. Structural elucidation of isolated enzymatic products by NMR and MS analyses proved unequivocally that the prenylation was regioselective at position C4 of the indole ring in the presence of dimethylallyl diphosphate. Determination of the kinetic parameters revealed that L-tryptophan was accepted as the best substrate by the enzyme, followed by 5-,6-,7-methyltryptophan and L-abrine. The enzymatic rate constant (k(cat) K(m) (-1)) of nine selected substrates were found to be about 1.0 to 6.5 % of that for L-tryptophan. Overnight incubation with eight substances showed that the conversion ratio to their prenylated derivatives was in the range 32.5 to 99.7 %. This provides evidence that 4-dimethylallylated indole derivatives can be produced by chemoenzymatic synthesis with FgaPT2.     

CdpNPT, an N-prenyltransferase from Aspergillus fumigatus: overproduction, purification and biochemical characterisation

A putative prenyltransferase gene, cdpNPT, was identified in the genome sequence of Aspergillus fumigatus by a homology search by using known prenyltransferases and sequence analysis. CdpNPT consists of 440 amino acids and has a molecular mass of about 50 kDa. The coding sequence of cdpNPT was cloned in pQE60 and overexpressed in E. coli. The soluble His(6)-fusion CdpNPT was purified to near homogeneity and characterised biochemically. The enzyme showed broad substrate specificity towards aromatic substrates and was found to catalyse the prenylation of tryptophan-containing cyclic dipeptides at N1 of the indole moieties in the presence of dimethylallyl diphosphate (DMAPP); geranyl diphosphate was not accepted as prenyl donor. The structures of the enzymatic products were elucidated by NMR and MS analysis. The K(m) value for DMAPP was determined to be 650 microM. Due to substrate inhibition, K(m) values could not be obtained for the aromatic substrates. CdpNPT does not need divalent metal ions for its enzymatic reaction, although Ca(2+) enhances the reaction velocity by up to the threefold. CdpNPT is the first N-prenyltransferase that has been purified and characterised in a homogenous form after heterologous overproduction. Interestingly, it shows significant sequence similarity to other indole prenyltransferases that catalyse the formation of C--C bonds.     

An enzyme catalyzing O-prenylation of the glucose moiety of fusicoccin A, a diterpene glucoside produced by the fungus Phomopsis amygdali

Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane‐bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant‐pathogenic fungus Phomopsis amygdali and has a unique O‐prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6′‐hydroxy group of the glucose moiety of FC A to yield an O‐prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 °C, respectively. K_m values were calculated as 0.49±0.037 μM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3±0.63 μM for DMAPP, with a k_cat of 55.3±3.3×10^?3 s. The enzyme did not act on representative substrates of the above‐mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.     

Geranylation of Cyclic Dipeptides by the Dimethylallyl Transferase AnaPT Resulting in a Shift of Prenylation Position on the Indole Ring

The dimethylallyl transferase AnaPT from Neosartorya fischeri is involved in the biosynthesis of acetylaszonalenin and catalyses the regioselective and stereospecific C3α‐prenylation of (R)‐benzodiazepinedione in the presence of dimethylallyl diphosphate. This enzyme also converts several tryptophan‐containing cyclic dipeptides to C3α‐prenylated indolines. In this study, we demonstrate the geranylation of (R)‐benzodiazepinedione and five other cyclic dipeptides by AnaPT in the presence of geranyl diphosphate (GPP). Interestingly, structure elucidation by NMR and MS analyses revealed that, with GPP, the geranyl moiety is attached to C‐6 or C‐7 rather than C‐3 of the indole ring of the enzyme products. For (R)‐benzodiazepinedione, one dominant C6‐geranylated derivative was obtained, whereas the other five substrates yielded both C6‐ and C7‐geranylated products. Neither acceptance of GPP by a dimethylallyl transferase from the dimethylallyltryptophan synthase superfamily, nor the alkylation shift from C‐3 to the benzene ring of the indole nucleus has been reported previously.     

Tryptophan C5‐, C6‐ and C7‐Prenylating Enzymes Displaying a Preference for C‐6 of the Indole Ring in the Presence of Unnatural Dimethylallyl Diphosphate Analogues

The behavior of four dimethylallyltryptophan synthases (DMATSs) (5‐DMATS and 5‐DMATS_Sc as tryptophan C5‐prenyltransferases, and 6‐DMATS_Sa and 6‐DMATS_Sv as C6‐prenyltransferases) and one L ‐tyrosine prenyltransferase with a tryptophan C7‐prenyltransferase activity was investigated in the presence of two unnatural alkyl donors (methylallyl and 2‐pentenyl diphosphate) and one benzyl donor (benzyl diphosphate). Detailed biochemical investigations revealed the acceptance of these dimethylallyl diphosphate (DMAPP) analogues by all tested enzymes with different relative activities. Enzyme products with the allyl or benzyl moiety attached to different positions were identified in the reaction mixtures, whereby C‐6 alkylated or benzylated L ‐tryptophan was found as one of the main products. This observation demonstrates a preference of the five prenyltransferases toward C‐6 of the indole ring in the presence of unnatural DMAPP derivatives. Molecular dynamics simulation experiments with a homologous model of 5‐DMATS explained well its reactions with methylallyl and 2‐pentenyl diphosphate. Furthermore this study expands significantly the potential usage of tryptophan prenylating enzymes as biocatalysts for Friedel–Crafts alkylation.

     

Evidence for the combined participation of a C10 and a C15 precursor in the biosynthesis of moenocinol, the lipid part of the moenomycin antibiotics

Upon feeding of [2-(13)C,4-(2)H]-1-deoxy-D-xylulose to Streptomyces ghanaensis, the deuterium label was retained exclusively at positions C-7 and C-17 in the moenocinol part of the moenomycin antibiotics. This result vindicates the hypothesis that the C(25) structure of moenocinol is assembled from a C(10) and a C(15) precursor, each of which requires for its formation the involvement of a dimethylallyl diphosphate starter unit.     

AstPT Catalyses both Reverse N1‐ and Regular C2 Prenylation of a Methylated Bisindolyl Benzoquinone

Prenylated bisindolyl benzoquinones exhibit interesting biological activities, such as antidiabetic or anti‐HIV activities. A number of these compounds, including asterriquinones, have been isolated from Aspergillus terreus. In this study, we identified two putative genes by genome mining, ATEG_09980 and ATEG_00702, which share high sequence similarity with the known bisindolyl benzoquinone prenyltransferase TdiB from Aspergillus nidulans. The coding sequences were cloned and overexpressed in E. coli. The overproduced recombinant proteins were purified to near homogeneity and used for enzyme assays with asterriquinone D in the presence of dimethylallyl diphosphate. HPLC analysis showed that product formation was only detected in enzyme assays with EAU29429 encoded by ATEG_09980, not in those with EAU39348 encoded by ATEG_00702. Product isolation and structure elucidation by NMR and MS analyses led to identification of N1‐reversely and C2‐regularly monoprenylated derivatives, as well as N1′,N1′′reversely, N1′‐reversely, C2′′‐regularly diprenylated derivatives. This proved that EAU29429 functions as an asterriquinone prenyltransferase (AstPT) and indicated the involvement of EAU29429 rather than EAU39348 in the biosynthesis of methylated asterriquinones. Furthermore, incubation of monoprenylated enzyme products with AstPT resulted in the formation of the diprenylated derivatives.     

Actions of Tryptophan Prenyltransferases Toward Fumiquinazolines and their Potential Application for the Generation of Prenylated Derivatives by Combining Chemical and Chemoenzymatic Syntheses

D i m ethyl a llyl t ryptophan s ynthases (DMATSs) catalyze regiospecific transfer reactions of a prenyl moiety from dimethylallyl diphosphate to various positions of the indole ring of tryptophan. For example, FgaPT2, 5‐DMATS, and 7‐DMATS from Aspergillus fungi catalyze tryptophan prenylation at C‐4, C‐5, and C‐7, while 5‐DMATS_Sc and 6‐DMATS_Sa from Streptomyces strains are tryptophan C‐5 and C‐6 prenyltransferases, respectively. In this study, our objective is to demonstrate the possibility of producing prenylated analogues of ardeemin fumiquinazoline (FQ) ( 1a ), a precursor of the m ulti d rug r esistance (MDR) export pump inhibitor ardeemin, by using DMATSs. All ardeemin FQ stereoisomers were chemically synthesized and used as substrates for enzyme assays with DMATSs. Biochemical investigations revealed different features of these enzymes toward ardeemin FQ analogues, regarding activity and prenylation position. Isolation and structural elucidation showed that 7‐DMATS catalyzed mainly regiospecific prenylation at C‐7, which is also observed for its natural substrate L ‐tryptophan. Up to four prenylated derivatives were identified in the reaction mixtures of other enzymes. In total, 18 new prenylated ardeemin FQ analogues were obtained in this study. Molecular modelling of ardeemin FQ analogues with the crystal structure of FgaPT2 led to the identification of two potential amino acid residues for guidance of the prenylation position. The two generated mutants FgaPT2_M328L and FgaPT2_Y398F showed significant prenylation shifts with 1a .

     

One Substrate – Seven Products with Different Prenylation Positions in One‐Step Reactions: Prenyltransferases Make it Possible

Prenylated indole alkaloids derived from L ‐tryptophan are widely distributed in nature and show diverse biological and pharmacological activities, usually distinct from their non‐prenylated precursors. Prenyltransferases catalyze the transfer reactions of prenyl moieties onto the indole nucleus and contribute largely to the structural diversity of these compounds. In this study, we demonstrate the acceptance of cyclo‐L ‐homotryptophan‐D ‐valine, an unnatural cyclic dipeptide, by eight prenyltransferases of the dimethylallyltryptophan synthase superfamily. Seven products with one prenyl moiety at each position of the indole nucleus and one diprenylated derivative were isolated from enzyme assays of cyclo‐L ‐homotryptophan‐D ‐valine with dimethylallyl diphosphate. To the best of our knowledge, this is the first report for production of seven monoprenylated products from one substrate by one‐step reactions.

     

New Insights into the Biosynthesis of Prenylated Xanthones: Xptb from Aspergillus nidulans Catalyses an O‐Prenylation of Xanthones

Gene‐inactivation experiments have indicated that the putative prenyltransferase XptB from Aspergillus nidulans was likely to be responsible for the prenylation of 1,7‐dihydroxy‐6‐methyl‐8‐hydroxymethylxanthone. Recently, it was suggested that this enzyme might also accept as substrate the benzophenone arugosin H, which is assumed to be a precursor of prenylated xanthones. In this study, five benzophenones and ten xanthones were incubated with purified recombinant XptB in the presence of dimethylallyl diphosphate (DMAPP). XptB accepted four xanthones as substrates, including the proposed natural substrate, and catalysed regiospecific O‐prenylations at C‐7 of the xanthone core. K_m values in the range of 0.081–1.1 mM and turnover numbers (k_cat) between 0.02 and 0.5 s^?1 were determined for the accepted xanthones. The kinetic parameters for DMAPP were found to be 0.024 mM (K_m) and 0.13 s^?1 (k_cat). Arugosin H was not accepted by XptB under the tested conditions. XptB was relatively specific towards its prenyl donor and did not accept geranyl or farnesyl diphosphate as substrate. Mn²⁺ and Co²⁺ strongly enhanced XptB activity (up to eightfold); this has not been reported before for prenyltransferases of the DMATS superfamily.     

AuaA, a membrane-bound farnesyltransferase from Stigmatella aurantiaca, catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the biosynthesis of aurachins

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA-auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. (1) H NMR and HR-EI-MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. K(M) values of 43 and 270 μM were determined for FPP and 2-methyl-4-hydroxyquinoline, respectively. Like other known membrane-bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg(2+) , Mn(2+) and Co(2+) , although no typical (N/D)DXXD binding motif was found in the sequence.     

Potential Rearrangements in the Reaction Catalyzed by the Indole Prenyltransferase FtmPT1

The indole prenyltransferase FtmPT1 catalyzes the C‐2 normal prenylation of brevianamide F (cyclo‐L ‐Trp‐L ‐Pro) to give tryprostatin B. A previous structural analysis and studies with alternate substrates suggest that the reaction might not proceed through a direct C‐2 attack, but could involve a C‐3 prenylation followed by a rearrangement. In this work we investigated the reactivity of FtmPT1 with tryptophan, 5‐hydroxybrevianamide, and 2‐methylbrevianamide, and isolated products that had been reverse prenylated at C‐3 and normal prenylated at N‐1, C‐3, or C‐4. The formation of these products can be rationalized through mechanisms involving either an initial C‐3 normal or C‐3 reverse prenylation. In addition, we demonstrate that a C‐3 reverse prenylated indole can undergo a nonenzymatic aza‐Cope rearrangement at 37 °C to give an N‐1 normal prenylated product. Together, these studies broaden the known product scope of this interesting catalyst and suggest that alternative mechanisms might be operating.     

A Membrane‐Bound Prenyltransferase Catalyzes the O‐Prenylation of 1,6‐Dihydroxyphenazine in the Marine Bacterium Streptomyces sp. CNQ‐509

Streptomyces sp. CNQ‐509 produces the rare O‐prenylated phenazines marinophenazines A and B. To identify the enzyme catalyzing the O‐prenyl transfer in marinophenazine biosynthesis, we sequenced the genome of S. sp. CNQ‐509. This led to the identification of two genomic loci harboring putative phenazine biosynthesis genes. The first locus contains orthologues for all seven genes involved in phenazine‐1‐carboxylic acid biosynthesis in pseudomonads. The second locus contains two known phenazine biosynthesis genes and a putative prenyltransferase gene termed cnqPT1. cnqPT1 codes for a membrane protein with sequence similarity to the prenyltransferase UbiA of ubiquinone biosynthesis. The enzyme CnqPT1 was identified as a 1,6‐dihydroxyphenazine geranyltransferase, which catalyzes the C?O bond formation between C‐1 of the geranyl moiety and O‐6 of the phenazine scaffold. CnqPT1 is the first example of a prenyltransferase catalyzing O‐prenyl transfer to a phenazine.     

Characterization of a single gene cluster responsible for methylpendolmycin and pendolmycin biosynthesis in the deep sea bacterium Marinactinospora thermotolerans

The nine-membered indolactam antibiotics belong to a small group of antibiotics showing broad biological activities. However, the in vivo genetic engineering of compounds of this type has not been performed. Here we report the identification of a single gene cluster responsible for the biosynthesis of methylpendolmycin and pendolmycin, two members of this family of antibiotics, from the deep sea bacterium Marinactinospora thermotolerans SCSIO 00652. Bioinformatics analysis and gene inactivation, coupled with metabolite characterization, reveal that MpnB, a nonribosomal peptide synthetase, MpnC, a cytochrome P450, and MpnD, a prenyltransferase, are sufficient to catalyze the biosynthesis of the two antibiotics from L-Ile (or L-Val), L-Trp, and methionine. MpnD is the first identified enzyme that transfers a C5 prenyl unit in a reverse manner to the C-7 position of a Trp-derived natural product.     

Characterization of the biosynthesis gene cluster for alkyl-O-dihydrogeranyl-methoxyhydroquinones in Actinoplanes missouriensis

A polyketide biosynthesis gene cluster (agq) was found on the genome of a rare actinomycete, Actinoplanes missouriensis. Streptomyces lividans expressing agqA encoding a type III polyketide synthase produced alkylresorcinols mainly from C(16-17) fatty acids. Heterologous expression of the agq genes in S. lividans indicated the function of cognate polyketide modification enzymes; a monooxygenase AgqB hydroxylates the alkylresorcinols to yield 6-alkyl-2-hydroxyhydroquinones, a methyltransferase AgqC catalyzes O-methylation of the alkyl-hydroxyhydroquinones to yield 6-alkyl-2-methoxyhydroquinones, and a UbiA-like prenyltransferase AgqD attaches a prenyl group to the C-4 hydroxy group of the alkyl-methoxyhydroquinones to yield 6-alkyl-4-O-geranyl-2-methoxyhydroquinones and 6-alkyl-4-O-dihydrofarnesyl-2-methoxyhydroquinones derived from C(16-17) fatty acids. In contrast, A. missouriensis was found to produce 6-alkyl-4-O-dihydrogeranyl-2-methoxyhydroquinones derived from C(16-18) fatty acids by the function of the agq gene cluster. All of these prenylated phenolic lipids were novel compounds.     

Taxonomic implication of sterol composition in the genusChlorella

Thirty-five isolates of the genusChlorella were grown under standardized conditions and analyzed for sterol composition. Six different sterol synthetic patterns were found. Two patterns were characterized by nuclear unsaturation at C-5 and one type by nuclear unsaturation at C-7. The remaining isolates all synthesize sterols with a diunsaturated nucleus (C-5+C-7) with various modifications. Subtype 1 synthesizes only C₂₈ sterols, subtype 2 produces C₂₈ and C₂₉ sterols, and subtype 3 (1 isolate) produces C₂₈ sterols with nuclear unsaturation at C-5+C-8 in addition to the Δ^5,7 C₂₈ sterols seen in the first 2 subtypes. Comparison of the sterol composition of strains shared in common with taxonomic researchers showed good correlation with the taxonomic scheme of Fott and Novakova which has been confirmed by Kessler and coworkers using other biochemical markers. Scientific article no. A-3030, contribution no. 6093 of the Maryland Agricultural Experiment Station.     

Extension of the Terpene Chemical Space: the Very First Biosynthetic Steps

The structural diversity of terpenes is particularly notable and many studies are carried out to increase it further. In the terpene biosynthetic pathway this diversity is accessible from only two common precursors, i. e. isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Methods recently developed (e. g. the Terpene Mini Path) have allowed DMAPP and IPP to be obtained from a two-step enzymatic conversion of industrially available isopentenol (IOH) and dimethylallyl alcohol (DMAOH) into their corresponding diphosphates. Easily available IOH and DMAOH analogues then offer quick access to modified terpenoids thus avoiding the tedious chemical synthesis of unnatural diphosphates. The aim of this minireview is to cover the literature devoted to the use of these analogues for widening the accessible terpene chemical space.     

Identification of the Verruculogen Prenyltransferase FtmPT3 by a Combination of Chemical,Bioinformatic and Biochemical Approaches

Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin‐type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O‐prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His₈‐FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. K_M values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 μM , respectively. Average turnover number (k_cat) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s^?1. FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13‐dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.