Increasing structure diversity of prenylated diketopiperazine derivatives by using a 4-dimethylallyltryptophan synthase

To create structural diversity of prenylated diketopiperazine derivatives, acceptance of cyclic dipeptides was tested using FgaPT2, a prenyltransferase from Aspergillus fumigatus, which catalyses the conversion of l-tryptophan to 4-dimethylallyl-l-tryptophan. It could be shown that seven tryptophan-containing cyclic dipeptides were accepted by FgaPT2 at high protein concentrations and regiospecifically converted to their C4 prenylated derivatives. The structures of the enzymatic products were elucidated by NMR and LC-MS analyses. This substrate promiscuity of a dimethylallyltryptophan synthase towards cyclic dipeptides increases the potential of the fungal indole prenyltransferases as tools for the production of biologically active compounds. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrolysis of cyclic nucleotides by a purified cGMP-stimulated phosphodiesterase: structural requirements for hydrolysis

Hydrolysis of cyclic AMP and cyclic GMP analogues by a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue was investigated by reversed-phase HPLC. The results indicate that both a negative charge and an equatorial oxygen atom located at the cyclic phosphate residue are absolute requirements for the process of hydrolysis. Other substituents only gradually decreased the apparent hydrolytic activity. C-8-substituted derivatives were generally poor substrates due to the limited ability of these compounds to rotate freely around the glycosidic bond. While C-6- and 0-2'-substituted analogues carrying bulky substituents were also poorly hydrolysed, all other derivatives, including different C-2-, C-6-, 0-3'- and 0-5'-modified cyclic nucleotides, were good substrates. We consistently observed that cyclic GMP and cyclic GMP analogues were better hydrolysed than the corresponding cyclic AMP analogues. Hydrolysis was correlated with neither the hydrogen bond donor/acceptor abilities nor the hydrophobicity of selected cyclic nucleotide analogues. Based on quantum-chemical calculations of the size and direction of the dipole moments of different purine bases, we propose that the polarization of inducible amino acid side-chains within the binding site is involved in the differential binding of adenine-derived and guanine-derived nucleotides. However, the size of the dipole moment alone is not sufficient to explain the observed cGMP-preference. Rather, the direction of the polarization power relative to the other molecular structures involved in binding and hydrolysis seems to be the molecular mechanism by which the enzyme is able to discriminate between cAMP- and cGMP-like structures.     

Prenylation at the indole ring leads to a significant increase of cytotoxicity of tryptophan-containing cyclic dipeptides

Fourteen tryptophan-containing cyclic dipeptides 1a-14a, including all four stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala, were converted to their C2-regularly prenylated derivatives 1b-14b in the presence of dimethylallyl diphosphate by using the purified recombinant FtmPT1 as catalyst. The enzyme products were isolated on HPLC in preparative scales and their structures were elucidated by NMR and MS analyses. The cytotoxic effects of the prenylated products and their substrates were tested with human leukemia K562 and ovarian cancer A2780 sens and A2780 CisR cell lines. Preliminary results have been clearly shown that prenylation at C2 led to a significant increase of the cytotoxicity of the tested cyclic dipeptides in all the 14 cases. The second amino acid and the stereochemistry of tryptophan moiety of the cyclic dipeptides showed less influence on the cytotoxicity of the tested compounds.     

Biochemical characterization of indole prenyltransferases: filling the last gap of prenylation positions by a 5-dimethylallyltryptophan synthase from Aspergillus clavatus

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.     

Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products

A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from various fungi. These genes belong to different gene clusters and are involved in the biosynthesis of secondary metabolites. Ten of them were cloned and overexpressed in Escherichia coli and Saccharomyces cerevisiae and proven to be soluble proteins. They catalyse different prenyl transfer reactions onto indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. These enzymes showed broad substrate specificities towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by several prenyltransferases as substrates and converted to prenylated derivatives. This feature of substrate flexibility was successfully used for regiospecific and stereospecific synthesis of different indole derivatives.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Identification of a brevianamide F reverse prenyltransferase BrePT from Aspergillus versicolor with a broad substrate specificity towards tryptophan-containing cyclic dipeptides

A putative brevianamide F reverse prenyltransferase gene brePT was amplified from Aspergillus versicolor NRRL573 by using primers deduced from its orthologue notF in Aspergillus sp. MF297-2 and overexpressed in Escherichia coli. The soluble His-tagged protein BrePT was purified to near homogeneity and assayed with tryptophan-containing cyclic dipeptides in the presence of dimethylallyl diphosphate. BrePT showed much higher flexibility towards its aromatic substrates than NotF and accepted all of the 14 tested tryptophan-containing cyclic dipeptides. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific reverse prenylation at C2 of the indole nucleus. K _M values of BrePT were determined for its putative substrates brevianamide F and DMAPP at 32 and 98 μM, respectively. Average turnover number (k _cat) at 0.4 s^?1 was calculated from kinetic data of brevianamide F and DMAPP. K _M values in the range of 0.082–2.9 mM and k _cat values from 0.003 to 0.15 s^?1 were determined for other 11 cyclic dipeptides. Similar to known fungal indole prenyltransferases, BrePT did not accept geranyl or farnesyl diphosphate as prenyl donor for its prenylation.     

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.     

Structural and functional analysis of bacterial flavin-containing monooxygenase reveals its ping-pong-type reaction mechanism

A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP(+) remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP(+), and a mutant bFMO(Y207S), which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP(+) and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP(+) revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMO(Y207S) exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole.     

Indole melatonin agonists and antagonists derived by shifting the melatonin side chain from the C-3 to the N-1 or to the C-2 indole position

This article reviews our efforts in the development of indole melatonin (MLT) agonist and antagonist compounds. Evidence is presented which indicates that high-affinity melatonergic agonists were obtained by shifting the MLT amido side chain from the C-3 to the N-1 indole position. Conversely, by moving the side chain from the C-3 to the C-2 indole position it is possible to produce MLT antagonist compounds.     

Mechanism of 3-methylaspartase probed using deuterium and solvent isotope effects and active-site directed reagents: identification of an essential cysteine residue.

The mechanism of the L-threo-3-methylaspartate ammonia-lyase (EC 4.3.1.2) reaction has been probed using deuterium and solvent isotope effects with three different substrates, (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid. Each substrate appears to form a covalent adduct with the enzyme through the amination of a dehydroalanine (DehydAla-173) residue. The true substrates are N-protonated and at low pH, the alkylammonium groups are deprotonated internally in a closed solvent-excluded pocket after K+ ion, an essential cofactor, has become bound to the enzyme. At high pH, the amino groups of the substrates are able to react with the dehydroalanine residue prior to K+ ion binding. This property of the system gives rise to complex kinetics at pH 9.0 or greater and causes the formation of dead-end complexes which lack Mg2+ ion, another essential cofactor. The enzyme-substrate adduct is subsequently deaminated in two elimination processes. Hydrazines act as alternative substrates in the reverse reaction direction in the presence of fumaric acid derivatives, but cause irreversible inhibition in their absence. Borohydride and cyanide are not inhibitors. N-Ethylmaleimide also irreversibly inactivates the enzyme and labels residue Cys-361. The inactivation process is enhanced in the presence of cofactor Mg2+ ions and Cys-361 appears to serve as a base for the removal of the C-3 proton from the natural substrate, (2S,3S)-3-methylaspartic acid. The dehydroalanine residue appears to be protected in the resting form of the enzyme by generation of an internal thioether cross-link. The binding of the substrate and K+ ion appear to cause a conformational change which requires hydroxide ion. This is linked to reversal of the thioether protection step and generation of the base for substrate deprotonation at C-3. The deamination reaction displays high reverse reaction commitments and independent evidence from primary deuterium isotope effect data indicates that a thiolate acts as the base for deprotonation at C-3.     

Enzymatic conversion-based method for screening cyclic dipeptide-producing microbes

We developed a method for screening cyclic dipeptide-producing microbes by enzymatic conversion. In this method, cyclic dipeptides are detected by the combination of: (i) conversion of cyclic dipeptides to dehydro cyclic dipeptides by cyclo(Leu-Phe) oxidase and (ii) detection of the dehydro derivative by UV spectrophotometry using TLC or HPLC analysis based on the absorbance change caused by the conversion. Using this method, the actinomycete strain A8 was isolated as a cyclic dipeptide-producing strain. The cyclic dipeptides were purified from the microbial extract by enzymatic detection-guided fractionation, and their structures were determined to be cyclo(L-Phe-L-Pro) and cyclo(L-Pro-L-Tyr) by spectroscopic methods.     

Regio-selective prenylation of flavonoids by plant cell suspension cultures of Cudrania tricuspidata and Morus alba

The cell suspension cultures of Cudrania tricuspidata could regio-selectively prenylate chrysin (1) at C-8, while the cell suspension cultures of Morus alba could regio-selectively prenylate genistein (2), sophoricoside (3) and diosmetin (4) at C-6. Eight products (5–12) were isolated, and five of them (8–12) were new compounds. Additionally, the bioconversion of 1–4 using microsomes of the cell cultures was performed, and the results showed that the bioconversion patterns were identical to those using cell cultures. These investigations would provide an approach to the selective prenylation and structural diversification of flavonoids.     

Parallel interrogation of covalent intermediates in the biosynthesis of gramicidin S using high-resolution mass spectrometry

For determination of multiple covalent intermediates bound to the ultra-large enzymes responsible for biosynthesis via nonribosomal peptide synthesis, mass spectrometry (MS) is a promising method to provide new mechanistic insight. Application of a quadrupole-Fourier-transform instrument (Q-FTMS) for direct analysis of aminoacyl intermediates is demonstrated for the first two modules (127 and 120 kDa) involved in the nonribosomal synthesis of gramicidin S. Cyanogen bromide digestions of recombinant proteins afforded detection of two active site peptides (both ~13 kDa) that provided direct evidence for modules copurifying with their preferred amino acid substrates. Given the ability to detect multiple covalent intermediates in tandem, a competition experiment among several nonnatural substrates in parallel was performed using the first module. This defined mixture of acyl-enzyme intermediates was used to probe the selectivity of the condensation step producing a diversity of noncognate dipeptides on the second module.     

A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

P450 monooxygenases from microorganisms, similar to those of eukaryotic mitochondria, display a rather narrow substrate specificity. For native P450 BM-3, no other substrates than fatty acids or an indolyl-fatty acid derivative have been reported (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537-544). We thus were quite surprised to observe, in the course of our investigations on the rational evolution of this enzyme towards mutants, capable of hydroxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87Val, Leu188-Gln, Ala74Gly, BM-3 mutant) could efficiently hydroxylate indole, leading to the formation of indigo and indirubin (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Indole is not oxidized by the wild-type enzyme; it lacks the carboxylate group by which the proper fatty acid substrates are supposed to be bound at the active site of the native enzyme, via hydrogen bonds to the charged amino acid residues Arg47 and Tyr51. Our attempts to predict the putative binding mode of indole to P450 BM-3 or the triple mutant by molecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigated in a preliminary, but systematic manner several alkanes, alicyclic, aromatic, and heterocyclic compounds, all of which are unaffected by the native enzyme, for their potential as substrates. We here report that this triple mutant indeed is capable to hydroxylate a respectable range of other substrates, all of which bear little or no resemblance to the fatty acid substrates of the native enzyme.     

Activity of fluoro and deoxy analogues of glycerol as substrates and inhibitors of glycerol kinase

Analogues of glycerol in which each of the three hydroxy groups is successively replaced by fluorine or hydrogen have been examined as substrates or inhibitors of glycerol kinase (Candida mycoderma) to assess the ability of fluorine to mimic a substrate hydroxy group in enzyme-analogue interactions. The four diols resulting from replacement of the hydroxy groups at C-1 or C-2 of sn-glycerol by fluorine or hydrogen are weak substrates. Similar substitution of the C-3 hydroxy group gives compounds which act as competitive inhibitors of glycerol or dihydroxyacetone phosphorylation but show no activity as substrates. Comparison of the steady-state kinetic parameters of the corresponding analogues shows that replacement of a hydroxy group by either fluorine or hydrogen leads to compounds with similar activity in this system. A convenient synthesis of (+)-propane-1,2-diol is described.     

Regioselectivity of 7-O-methyltransferase of poplar to flavones

POMT-7, an O-methyltransferase from poplar (Populus deltoids) was used to modify a variety of flavonoid compounds. POMT-7 was able to transfer a methyl group to several flavonoids containing a C-7 hydroxyl group. However, POMT-7 showed a higher affinity toward flavonol and flavone such as apigenin, kaempferol, luteolin, and quercetin than flavanone and isoflavone. Based on comparison of HPLC retention times with authentic compounds and corresponding nuclear magnetic resonance spectroscopy data, the methylation position of the reaction products was determined to be at the hydroxyl group of C-7. Biotransformation kinetics indicated that the enzyme converted more than 80% of the apigenin, kaempferol, luteolin and quercetin substrates, which were added at concentration of 70 microM, into corresponding 7-methoxy compounds within 24 h.     

Rational design and synthesis of topoisomerase I and II inhibitors based on oleanolic acid moiety for new anti-cancer drugs

Semisynthetic reactions were conducted on oleanolic acid, a common plant-derived oleanane-type triterpene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and tested for their topoisomerase I and IIα inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-13, and C-17. Nine of the synthesized compounds were identified as new compounds. The structures of these compounds were confirmed by spectroscopic methods (1D, 2D NMR and MS). Five oleanolic acid analogues (S2, S3, S5, S7 and S9) showed higher activity than camptothecin (CPT) in the topoisomerase I DNA relaxation assay. Four oleanolic acid analogues (S2, S3, S5 and S6) showed higher activity than etoposide in a topoisomerase II assay. The results indicated that the C12–C13 double bond of the oleanolic acid skeleton is important for the inhibitory activity against both types of topoisomerases, while insertion of a longer chain at either position 3 or 17 increases the activity against topoisomerases by various degrees. Some of the synthesized compounds act as dual inhibitors for both topoisomerase I and IIα.     

gamma-Glutamyl dipeptides in Petiveria alliacea

Three gamma-glutamyl dipeptides have been isolated from Petiveria alliacea L. roots. These dipeptides include (S(C2)R(C7))-gamma-glutamyl-S-benzylcysteine together with two diastereomeric sulfoxides, namely (S(C2)R(C7)R(S))- and (S(C2)R(C7)R(S))-gamma-glutamyl-S-benzylcysteine S-oxides (gamma-glutamyl-petiveriins A and B, respectively). Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy, and confirmed by comparison with authentic compounds obtained by synthesis.     

异戊烯基化吲哚类生物碱的化学酶合成

异戊烯基化吲哚类生物碱广泛存在于麦角菌、青霉菌和曲霉菌中,具有一定的药理学活性,与未异戊烯基化的前体在生物活性方面具有明显的差异.曲霉菌中的某些异戊烯基化吲哚类生物碱具有抗癌活性,如烟曲霉毒素C（fumitremorgin C）、tryprostatin B,但其天然产量低且不易分离,利用化学酶合成法可很容易地将前体转化为异戊烯基化吲哚类生物碱.异戊烯基转移酶FtmPT1对二甲丙烯基二磷酸（dimethylallyl diphosphate,DMAPP）具有专一性,但可以接受不同的芳香族底物.早期研究发现,FtmPT1能接受含色氨酸的不同环二肽为底物,但以cyclo-L-Trp-L-Tyr和cyclo-L-Trp-L-Phe为底物时,酶的相对活性很低,其产物量少,无法用于合成产物.本实验通过优化酶反应条件来提高其产量.将已构建的含ftmPT1的质粒在大肠杆菌中诱导表达,经Ni-NTA亲和柱纯化后用于酶反应.实验结果表明,通过增加酶量（终浓度2.8 μmol/L）、延长培养时间（37 ℃,24 h）,以cyclo-L-Trp-L-Tyr和cyclo-L-Trp-L-Phe为底物的酶反应产率分别达到49.3%和21.3%,产物经¹H^-NMR、¹H-¹H-COSY和ESI-MS鉴定,其结果与预期吻合.据检索,这2个化合物均为新化合物,分别命名为cyclo-C2-1′-DMA-L-Trp-L-Tyr和cyclo-C2-1′-DMA-L-Trp-L-Phe.