Reverse prenyltransferase in the biosynthesis of fumigaclavine C in Aspergillus fumigatus: gene expression, purification, and characterization of fumigaclavine C synthase FGAPT1

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.     

色氨酸酶重组基因表达及其酶法合成L-色氨酸

为实现色氨酸酶高效、低成本催化合成L-色氨酸,利用p ET30a为载体在宿主细胞E.coli BL21(DE3)中重组表达了产气肠杆菌(Enterobacter aerogenes)来源的色氨酸酶,以丙酮酸、吲哚和氨为底物,探究其酶学性质,考察了反应温度、起始p H、底物摩尔比对酶促反应的影响,并利用丙酮酸发酵液为底物酶法合成L-色氨酸。结果表明,色氨酸酶重组表达成功,色氨酸酶最佳反应条件为:温度35℃,起始p H=9.0,底物摩尔比n(吲哚)∶n(丙酮酸)=0.6∶1,底物丙酮酸浓度为0.17 mol/L。利用重组色氨酸酶全细胞催化100 m L浓度为0.57 mol/L丙酮酸发酵液,流加浓度为4.27 mol/L吲哚酒精溶液6.5 m L,反应28 h后,L-色氨酸浓度达0.25 mol/L,吲哚摩尔转化率达91.8%。     

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。     

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 .

     

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.     

Engineered RebH Halogenase Variants Demonstrating a Specificity Switch from Tryptophan towards Novel Indole Compounds

Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure-guided semi-rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated.     

Biochemical Investigations of Two 6‐DMATS Enzymes from Streptomyces Reveal New Features of L‐Tryptophan Prenyltransferases

Two putative prenyltransferase genes, SAML0654 and Strvi8510, were identified in Streptomyces ambofaciens and Streptomyces violaceusniger, respectively. Their deduced products share 63 % sequence identity. Biochemical investigations with recombinant proteins demonstrated that L ‐tryptophan and derivatives, including D ‐tryptophan, 4‐, 5‐, 6‐ and 7‐methyl‐dl ‐tryptophan, were well accepted by both enzymes in the presence of DMAPP. Structural elucidation of the isolated products revealed regiospecific prenylation at C‐6 of the indole ring and proved unequivocally the identification of two very similar 6‐dimethylallyltryptophan synthases (6‐DMATS). Detailed biochemical investigations with SAML0654 proved L ‐tryptophan to be the best substrate (K_m 18 μm, turnover 0.3 s^?1). Incubation with different prenyl donors showed that they also accepted GPP and catalyzed the same specific prenylation. Utilizing GPP as a prenyl donor has not been reported for tryptophan prenyltransferases previously. Both enzymes also catalyzed prenylation of some hydroxynaphthalenes; this has not previously been described for bacterial indole prenyltransferases. Interestingly, SAML0654 transferred prenyl moieties onto the unsubstituted ring of hydroxynaphthalenes.     

Structural Characterization and Extended Substrate Scope Analysis of Two Mg2+-Dependent O-Methyltransferases from Bacteria**

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized the enzymatic properties and substrate scope of two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids, and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether, and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering.     

Pipecolic Acid Hydroxylases: A Monophyletic Clade among cis‐Selective Bacterial Proline Hydroxylases that Discriminates l‐Proline

Proline hydroxylases are iron(II)/2‐oxoglutarate‐dependent enzymes that hydroxylate l ‐proline and derivatives, such as l pipecolic acid, which is the six‐membered‐ring homologue of l ‐proline. It has been established that there is a distinct group of conserved bacterial enzymes that hydroxylate l ‐pipecolic acid and trans‐3‐ and trans‐4‐methyl‐l ‐proline, but virtually no l ‐proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent l ‐proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp.; producer of the tetrapeptide antibiotic GE81112) and PiFa (from Frankia alni) overlaps that of proline hydroxylases, except for the nonacceptance of l ‐proline and smaller homologues. Distinct and conserved residues were determined for both types of enzymes. However, site‐directed mutagenesis in GetF did not yield variants that accepted l ‐proline; this suggested a complex interaction of several residues around the active site, which resulted in delicate changes in substrate specificity. This is supported by substrate docking in a homology model of GetF, which revealed an altered orientation for l ‐proline relative to that of preferred substrates.     

Kinetics of the enzymatic hydrolysis of lignocellulosic materials at different concentrations of the substrate

The kinetics of the enzymatic hydrolysis of two substrates—lignocellulosic materials from Miscanthus and oat hulls—in an acetate buffer is studied at different concentrations of the substrates. The substrates are obtained via single-step treatment with a dilute solution of nitric acid. The content of a nonhydrolyzable component—acid-insoluble lignin—for Miscanthus and oat hulls was 11 and 14%, respectively. A multi-enzyme composition of commercially available enzyme preparations CelloLux-A and BrewZyme BGX was used as a catalyst. It is shown that treatment with the nitric acid solution produces reactive substrates for the enzymatic hydrolysis. The innovative science of the results is confirmed by Russian patent 2533921. Kinetics of the enzymatic hydrolysis of these substrates in an acetate buffer can be described by a mathematical model based on a modified Michaelis–Menten equation. The main kinetic constants for both substrates are determined from the experimental data. The equilibrium concentrations of reducing substances (RSes) for the substrates are calculated from the initial substrate concentrations. It is found that within the studied range of substrate concentrations (33.3–120.0 g/L), the initial rate of enzymatic hydrolysis for the lignocellulosic material from oat hulls is higher than that for the lignocellulosic material from Miscanthus by 1 g/(L h). It is shown that the yield of RS depends of the initial concentration of the substrates: as the concentration rises from 33.3 to 120 g/L, the yield of RS falls 1.5–2.0 times, due to substrate inhibition. At low initial concentrations, the yields of RS are similar for the substrates from Miscanthus and oat hulls. When the initial concentration of the substrate reaches 120 g/L, the yield of reducing substances for the lignocellulosic material from Miscanthus is approximately 20% higher than that for the lignocellulosic material from oat hulls. The established dependences and the proposed mathematical model allow us to optimize the initial concentration of the substrate for efficient enzymatic hydrolysis.     

Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution

Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described. Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones. Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme. Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives. As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.     

Stepwise Post-glycosylation Modification of Sugar Moieties in Kanamycin Biosynthesis

Kanamycin A is the major 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotic produced by Streptomyces kanamyceticus. The 2DOS moiety is linked with 6-amino-6-deoxy-d -glucose (6ADG) at O-4 and 3-amino-3-deoxy-d -glucose at O-6. Because the 6ADG moiety is derived from d -glucosamine (GlcN), deamination at C-2 and introduction of C-6-NH₂ are required in the biosynthesis. A dehydrogenase, KanQ, and an aminotransferase, KanB, are presumed to be responsible for the introduction of C-6-NH₂, although the substrates have not been identified. Here, we examined the substrate specificity of KanQ to better understand the biosynthetic pathway. It was found that KanQ oxidized kanamycin C more efficiently than the 3′′-deamino derivative. Furthermore, the substrate specificity of an oxygenase, KanJ, that is responsible for deamination at C-2 of the GlcN moiety was examined, and the crystal structure of KanJ was determined. It was found that C-6-NH₂ is important for substrate recognition by KanJ. Thus, the modification of the GlcN moiety occurs after pseudo-trisaccharide formation, followed by the introduction of C-6-NH₂ by KanQ/KanB and deamination at C-2 by KanJ.     

Extensive Re‐Investigations of Pressure Effects in Rhodium‐ Catalyzed Asymmetric Hydrogenations

The catalytic hydrogenation of three pro‐chiral substrates methyl Z‐α‐acetamidocinnamate (MAC), methyl 2‐acetamidoacrylate (M‐Acrylate) and ethyl 4‐methyl‐3‐acetamido‐2‐propanoate (E‐EMAP) with rhodium precursors complexed with chiral diphosphines is reported at 1–30 bar hydrogen pressure. A library of 56 chiral diphosphines, including 23 BINAP derivatives, 7 JOSIPHOS, 5 BIPHEP, 3 DUPHOS derivatives, and 18 other ligands, was used. While it was generally accepted that high hydrogen pressure would result in lower ees, it is now demonstrated on a statistical basis that an equivalent distribution between beneficial and detrimental pressure effects on ee prevails and that the hydrogen pressure effect on enantioslectivity is not an isolated phenomenon since more than 33% of the reaction systems studied are strongly affected. In some case, the enantioselectivity can be improved up to 97% just by applying a higher hydrogen pressure. Extension of these conclusions to other non‐chiral reagents is proposed.     

A new route to 4-, 5-, and 6-indolecarboxylic acids

There are many indole derivatives bearing carboxyl or methoxycarbonyl groups on the benzene ring and these often have patent physiological activities. Synthesis of these compounds usually starts from indoles having a halogen group at the desired position on an indole ring. We now report a facile synthesis of 4-, 5-, and 6-indolecarboxylic acids from 2-bromoaniline derivatives ( 1 ). The synthetic route is shown in Figure 1.     

取代基物化参数用于2-苯基吲哚衍生物构效关系的研究

用取代基电性、立体性和疏水性物化参数对2-苯基吲哚衍生物进行了结构表征,并对化合物与雌激素受体相对亲和力进行定量结构活性相关(QSAR)研究。经逐步回归筛选变量后,所建多元线性回归方程的复相关系数R2及留一法交互检验相关系数R2cv分别为0.900和0.662。用预测集样本进行了外部预测,所得外部预测样本集复相关系数Re2xt和外部预测集交互检验Q2ext分别为0.896 6和0.897 4。模型结果显示:吲哚环1号N原子上立体效应强的取代基有利于亲和力的提高;而吲哚环1号N原子和3号C原子上电性强的取代基、2-苯基环上4′号位置有羟基取代等,均对化合物亲和力的提高不利。     

Asymmetric Friedel–Crafts Reaction of Indoles with Ethyl Trifluoropyruvate Using a Copper(I)‐Bisoxazolidine Catalyst

Bisoxazolidine 1 is an effective ligand in the copper(I)‐catalyzed Friedel–Crafts reaction of alkyl trifluoropyruvates and indoles. A range of ethyl 2‐(3′‐indolyl)‐3,3,3‐trifluoro‐2‐hydroxypropanoates was produced in up to 99% yield and 94% ee within 30 min to 4 h. The effect of temperature on conversion and enantioselectivity proved to be substrate specific and was optimized individually. Of particular interest is that this method tolerates the presence of substituents in various positions in the indole ring. Yields ranging from 90–97% and ee values between 90 and 94% were obtained at optimized temperatures with substrates carrying substituents in position 1 or 7.     

A novel method for the introduction of a methyl group into the furan ring of a 2,5-disubstituted C18 furanoid fatty estervia a malonic acid function

Furan ring opening with benzohydroxamic acid of methyl 9,12-epoxy-9,11-octadecadienoate gave a mixture of positional isomers of conjugated methyl 3-phenyl-1,4,2-dioxazolyl C₁₈-enone esters 6a,6b. Michael addition of diethyl malonate anion to the conjugated enone system of 6a,6b furnished the corresponding malonyl intermediates 7a,7b, which upon removal of the dioxazole ring by hydrolysis gave methyl 10- and 11-dicarbethoxymethyl-9,12-dioxooctadecanoate 8a,8b. Cyclization of the latter gave the trisubstituted C₁₈ furanoid fatty esters 9a,9b, containing the malonate ester function at the 3-/4-position of the furan ring. Base hydrolysis of compounds 9a,9b gave the corresponding tricarboxylic acid derivatives 10a,10b, which were esterified to the trimethyl esters 11a,11b in BF₃/MeOH. When a mixture of 9a,9b was refluxed with Na₂CO₃/MeOH, hydrolysis of the malonate ester function was followed by decarboxylation to yield a-CH₂COOH substituent at the 3-/4-position of the furan ring (12a, 12b). Esterification of the latter with BF₃/MeOH gave the corresponding methyl diester derivatives 13a,13b. When a mixture of tricarboxylic acids 10a,10b was heated at 160–180°C for 6 hr, exhaustive decarboxylation of malonic acid function furnished a methyl group at the 3-/4-position of the furan nucleus. Esterification of the decarboxylated product gave a mixture of trisubstituted furanoid compounds 14a,14b (overall yield 28%). The procedure constitutes a novel method for the introduction of a methyl groupvia a malonic acid group to the 3-/4-position of the furan ring of a 2,5-disubstituted C₁₈ furanoid fatty ester.     

Hydroxyalkoxy derivatives of oxamic acid: polyol substrates for polyurethane foams

BACKGROUND: The work reported aimed at obtaining hydroxyalkoxy derivatives containing oxalamidoester groups starting from oxamic acid (OA) substrate. These derivatives were then used for the synthesis of polyurethane foams. RESULTS: The hydroxyalkoxy derivatives were obtained by reaction of OA with 6 to 15 molar excess of ethylene carbonate (1,3‐dioxolane‐2‐one) or propylene carbonate (4‐methyl‐1,3‐dioxolane‐2‐one). The products obtained have good thermal stability and possess suitable physical properties for use as substrates for foamed polyurethanes. CONCLUSION: The rigid polyurethane foams obtained from the hydroxyalkoxy derivatives of OA possess enhanced thermal stability and good mechanical properties in comparison with traditional polyurethane foams. Copyright © 2009 Society of Chemical Industry     

Synthesis of derivatives of Naphtol AS containing polar groups and modification used for C.I. Pigment Red 57

The synthesis of derivatives of Naphtol AS containing polar groups (–COOH, –SO₃H) and factors affecting the reaction have been studied. Four derivatives of Naphtol AS containing –COOH or –SO₃H groups in the 3- or 4- position of the phenyl ring were synthesized. Derivatives of Naphtol AS as the second coupling component were used in mixed coupling and the effect of its added amount on the colour strength, hue, flowability and dispersing extent (in water) of pigments was studied.