A 7-dimethylallyl tryptophan synthase from a fungal Neosartorya sp.: Biochemical characterization and structural insight into the regioselective prenylation

A putative 7-dimethylallyl tryptophan synthase (DMATS) gene from a fungal Neosartorya sp. was cloned and overexpressed as a soluble His₆-fusion protein in Escherichia coli. The enzyme was found to catalyze the prenylation of l-tryptophan at the C7 position of the indole moiety in the presence of dimethylallyl diphosphate; thus, it functions as a 7-DMATS. In this study, we describe the biochemical characterization of 7-DMATS from Neosartorya sp., referred to as 7-DMATS^Neo, and the structural basis of the regioselective prenylation of l-tryptophan at the C7 position by comparison of the three-dimensional structural models of 7-DMATS^Neo with FgaPT2 (4-DMATS) from Aspergillus fumigatus.     

Enzymatic production of l-tryptophan from dl-5-indolylmethylhydantoin by Flavobacterium sp.

An enzymatic production of l-tryptophan from dl-5-indolylmethylhydantoin by the action of hydantoinase and carbamoylase has been investigated. A strain identified as (Flavobacterium) sp. I-3 isolated from soil was found to form l-tryptophan from dl-5-indolylmethylhydantoin. Cultural conditions for the formation of the l-tryptophan-forming activity were investigated, and the highest activity of 0.81 μmol min⁻¹of l-tryptophan formed per 1 ml of culture broth (hydantoinase, 3.6 μmol min⁻¹of N-carbamoyl-l-tryptophan formed per 1 ml of culture broth; carbamoylase, 0.92 μmol min⁻¹of l-tryptophan formed per 1 ml of culture broth) was obtained. These activities were found to be inducible and intracellular. Optimization of the parameters of the conversion reaction resulted in accumulation of 50 mg of l-tryptophan per 1 ml of cultural broth per day. The conversion yield from dl-5-indolylmethylhydantoin was about 100%. Accumulated l-tryptophan was readily isolated in pure form by ordinary procedures.     

Entrapment of l-tryptophan producing Escherichia coli in different matrices: activity of immobilized cells

Cells of Escherichia coli induced for l-tryptophan synthase [l-serine hydro-lyase (adding indole-glycerol-phosphate), EC 4.2.1.20] have been assayed in DMF and DMSO aqueous solvents as reaction medium. Up to 20% DMF/water, cells retained 90% of their tryptophan synthase activity. Concentrations of 20 mM indole, which did not inhibit this reactivity, could be reached with 5% DMF/water. Four matrices were compared for cell immobilization: polyacrylamide, foam particles of bovine seum albumin, alginate and κ-carrageenan. The best activity was retained with the latter matrix, and the preparations thus obtained allowed high productivity of l-tryptophan. Various systems of production of l-tryptophan with κ-carrageenan and DMF/water were studied.     

Effect of gene knockouts of l-tryptophan uptake system on the production of l-tryptophan in Escherichia coli

Uptake of l-tryptophan in Escherichia coli was carried out by three distinct permeases, Mtr, TnaB, and AroP, respectively. In the present study, the three genes of l-tryptophan uptake system were knocked out from an l-tryptophan-producing strain of E. coli, respectively. The knockout mutants all showed lower l-tryptophan uptake activities and higher l-tryptophan production than their parent. Among the three genes, the knockout of mtr was most critical for both l-tryptophan uptake and l-tryptophan production. The uptake activity of l-tryptophan of the mtr mutant was 1.5 nmol min^?1 (mg dry weight)^?1, which was decreased by 48% when compared to that of the parent; the production of l-tryptophan of the mtr mutant was 14.7 g/l, which was increased by 34% when compared to that of the parent. Furthermore, the physiological and fermentation characteristics caused by gene knockouts were also analyzed.     

模块化工程改造大肠杆菌生产L-色氨酸

L-色氨酸作为一种必需氨基酸,广泛应用于食品、饲料和医药等领域。目前,微生物法生产L-色氨酸存在转化率低等问题。为此,本研究通过敲除L-色氨酸操纵子阻遏蛋白(L-tryptophan operon repressor protein, trpR)、替换l-色氨酸弱化子(trpL)、引入抗反馈调节的aroG^fbr等,获得可积累11.80 g/L L-色氨酸的底盘菌株大肠杆菌(Escherichia coli)TRP3。在此基础上,将L-色氨酸合成途径分为中心代谢途径模块、莽草酸(shikimic acid, SA)途径至分支酸(chorismic acid, CHA)模块、分支酸至L-色氨酸模块,并借助启动子工程,通过平衡中心代谢途径模块、莽草酸途径至分支酸模块、分支酸至L-色氨酸模块,获得工程菌E.coli TRP9。在5 L发酵罐中,工程菌E.coli TRP9的L-色氨酸产量提升至36.08 g/L,糖酸转化率提升至18.55%,达到理论转化率的81.7%。本研究利用模块工程策略,构建了高产L-色氨酸生产菌株,为l-色氨酸的规模化生产奠定了良好的基础。     

Photoactivation of pseudomonad L-tryptophan oxygenase by electron ejection from its substrate, L-tryptophan

Chemically oxidized, catalytically inactive, pseudomonad l-tryptophan-2,3-dioxygenase (EC 1.13.1.12) can be photoactivated aerobically as well as anaerobically by light of wavelength less than 360 nm. The substrate, l-tryptophan, must be present for photoactivation to proceed. In these studies, a CCl₄ filter was used to block light of wavelength less than 265 nm, preventing photolysis of water and the concomitant production of H₂O₂ (known reductant of tryptophan oxygenase). Photoactivation is not inhibited by superoxide dismutase or formate and is only slightly inhibited by catalase. Nonsubstrate analogues of l-tryptophan, 5-fluorotryptophan (binds to the catalytic site), and α-methyltryptophan (binds to the allosteric site), separately or in concert, do not mediate photoactivation, while another substrate, 6-fluorotryptophan, can. Saturation of the allosteric site with α-methyltryptophan increases the extent of photoactivation in the presence of a nonsaturating level of l-tryptophan, indicating that photoactivation is dependent on the extent of saturation of the catalytic site by l-tryptophan. During the time course of photoactivation, catalytic activity increases faster than does the formation of ferroheme enzyme, indicating that the fully reduced enzyme, (ferroheme)₂(Cu⁺)₂, is formed from the fully oxidized enzyme, (ferriheme)₂(Cu²⁺)₂, subsequent to photoactivation. A significant amount of the half-reduced, catalytically active enzyme, (ferriheme)₂(Cu⁺)₂, exists during the time course of photoactivation. We propose that the mechanism by which electrons enter tryptophan oxygenase is via “electron ejection” [T. R. Hopkins and R. Lumry (1972) Photochem. Photobiol.15, 555–566] from a photoexcited l-tryptophan bound at, the catalvtic site.     

Inhibition of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase by β-carboline and indole derivatives

β-Carboline derivatives inhibited both indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase activities from various sources. Among them, norharman is most potent for both enzymes from mammalian sources. Kinetic studies revealed that norharman is uncompetitive (K_i = 0.12 mm) with l-tryptophan for rabbit intestinal indoleamine 2,3-dioxygenase, and linearly competitive (K_i = 0.29 mm) with l-tryptophan for mouse liver tryptophan 2,3-dioxygenase. In addition, some β-carbolines selectively inhibited one enzyme or the other. Pseudomonad tryptophan 2,3-dioxygenase was inhibited by a different spectrum of β-carbolines. Such a selective inhibition by the structure of substrate analogs is more evident by the use of indole derivatives. Indole-3-acetamide, indole-3-acetonitrile and indole-3-acrylic acid exhibited a potent inhibition for mammalian tryptophan 2,3-dioxygenase, while they moderately inhibited the pseudomonad enzyme. However, they showed no inhibition for indoleamine 2,3-dioxygenase. These results suggest the difference of the structures of the active sites among these enzymes from various sources.     

Development of tryptophan-modified human serum albumin columns for site-specific studies of drug–protein interactions by high-performance affinity chromatography

Human serum albumin (HSA) is one of the main proteins involved in the binding of drugs and small solutes in blood or serum. This study examined the changes in chromatographic properties that occur for immobilized HSA following the chemical modification of HSA's lone tryptophan residue (Trp-214). Trp-214 was reacted with o-nitrophenylsulfenyl chloride, followed by immobilization of the modified protein and normal HSA onto separate silica-based HPLC supports. The binding properties of the modified and normal HSA were then analyzed and compared by using frontal analysis and zonal elution experiments employing R/S-warfarin and l-tryptophan as probe compounds for the warfarin and indole binding regions of HSA. The modified HSA was found to have the same number of binding sites as normal HSA for R-warfarin and l-tryptophan but lower association equilibrium constants for these test solutes. Zonal elution studies with R- and S-warfarin on the modified HSA column demonstrated the importance of Trp-214 in determining the stereoselective binding of HSA for these agents. These studies also indicated that tryptophan modification can alter HSA-based separations for chiral solutes.     

Purification and properties of dimethylallylpyrophosphate: Tryptophan dimethylallyl transferase,the first enzyme of ergot alkaloid biosynthesis in Claviceps. sp. SD 58

Dimethylallylpyrophosphate:l-tryptophan dimethylallyltransferase (DMAT synthetase), the first pathway-specific enzyme of ergot alkaloid biosynthesis, has been isolated from mycelia of Claviceps sp., strain SD 58, and purified to apparent homogeneity. The enzyme reaction products were identified as l-4-(γ,γ-dimethylallyl)tryptophan and inorganic pyrophosphate. DMAT synthetase is a single subunit protein of molecular weight 70,000–73,000 and has an isoelectric point at pH 5.8. The enzyme is activated by Fe²⁺, Mg²⁺, and particularly Ca²⁺; K_m values for l-tryptophan and dimethylallylpyrophosphate were determined to be 0.067 and 0.2 mm, respectively. Kinetic analysis indicated that the DMAT synthetase reaction proceeds by a sequential rather than a ping-pong mechanism.     

L-tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo

The administration of l-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo. When l-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy-dl-tryptophan and indole administration in amounts equimolar to l-tryptophan also result in similar increases in activity whereas α-methyl-dl-tryptophan produces little or no increase.Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.     

Identification and biochemical characterization of a 5-dimethylallyl tryptophan synthase in Streptomyces coelicolor A3(2)

The genome sequencing of Streptomyces coelicolor A3(2) has lead to the identification of numerous cryptic gene clusters involved in the biosynthesis of secondary metabolites; throwing open the challenge of identifying the enzymatic functions that the gene clusters are associated with. In this work, we report the biochemical characterization of one such cryptic gene, SCO7467 from S. coelicolor A3(2), which is annotated as a prenyltransferase. Based on LC–MS and 2D-NMR studies, we show that SCO7467 acts as a 5-dimethylallyl tryptophan synthase (5-DMATS), and catalyzes the transfer of a dimethylallyl group to the C-5 position of the indole ring of l-tryptophan. The studies indicate that SCO7467 could be involved in the synthesis of C-5 prenylated indole alkaloids, which may exhibit unique pharmacological and biological properties.     

New indolesulfonamide derivatives targeting the colchicine site of tubulin: synthesis,anti-tumour activity,structure–activity relationships,and molecular modelling

Searching for improved indolesulfonamides with higher polarities, 45 new analogues with modifications on the sulfonamide nitrogen, the methoxyaniline, and/or the indole 3-position were synthesised. They show submicromolar to nanomolar antiproliferative IC₅₀ values against four human tumour cell lines and they are not P-glycoprotein substrates as their potencies against HeLa cells did not improve upon cotreatment with multidrug resistance (MDR) inhibitors. The compounds inhibit tubulin polymerisation in vitro and in cells, thus causing a mitotic arrest followed by apoptosis as shown by cell cycle distribution studies. Molecular modelling studies indicate binding at the colchicine site. Methylated sulfonamides were more potent than those with large and polar substitutions. Amide, formyl, or nitrile groups at the indole 3-position provided drug-like properties for reduced toxicity, with Polar Surface Areas (PSA) above a desirable 75 Ų. Nitriles 15 and 16 are potent polar analogues and represent an interesting class of new antimitotics.     

On the interaction between flavin-adenine rings and between flavin-indole rings by X-ray structural studies

Two crystal structures of 7,8-dimethylisoalloxazine-10-acetic acid:adenine-9-ylethylamine(1:1)heptahydrate and 7,8-dimethylisoalloxazine-10-acetic acid:l-tryptophan methylester(1:1)heptahydrate complexes were determined as models for the flavin-adenine and flavin-indole interactions, respectively. In the former complex, both molecules were connected by Hoogsteen-type hydrogen bonds between the pyrimidinoid portion of flavin and the adenine, in addition to the normal stacking of both aromatic rings. On the other hand, parallel stackings and intermolecular vertical spacings less than the normal van der Waals separation distance were observed between the flavin and indole rings of the latter complex, indicative of the π_D-π_A charge-transfer interaction in their ground states. Comparing with the X-ray findings of related complexes, we discussed the interaction modes between flavin and adenine rings and between flavin and indole rings.     

Molecular orbital study on the interaction between coenzymes and enzymes; NAD+ or NADH and dehydrogenases

The absorption band at 260 mμ of NAD⁺ shifts to 360 mg by interaction with GAPDH or its analogues. Two explanations have been given on this red shift; one is an addition of such nucleophilic residue as sulfhydryl group in the enzyme to the position four in nicotinamide nucleus of NAD⁺, and the other is the charge transfer from such aromatic amino acid as tryptophan to NAD⁺. In the present paper, possibility of the charge transfer from indole residue to NAD⁺ was investigated quantum chemically. Taking into account of the electric field due to the charges in the enzyme, the absorption band of the NAD⁺-enzyme complex at 360 mμ was explained as a charge transfer from indole nucleus to NAD⁺. The blue shift of the absorption band of NADH at 340 mμ was also explained by taking into account of the electric field and this supported the proposition of Kosower (1962a).Stacking of adenine nucleus with indole nucleus in the NAD⁺-enzyme complex was suggested from the NMR spectroscopic data. Our molecular orbital calculations predicted that the effects of adenine on spectral shifts were not significant.     

In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum

The purple chromobacterial pigment violacein arises by enzymatic oxidation and coupling of two molecules of l-tryptophan to give a rearranged pyrrolidone-containing scaffold in the final pigment. We have purified five contiguously encoded proteins VioA-E after expression in Escherichia coli and demonstrate the full 14-electron oxidation pathway to yield the final chromophore. The flavoenzyme VioA and the heme protein VioB work in conjunction to oxidize and dimerize l-tryptophan to a nascent product that can default to the off pathway metabolite chromopyrrolic acid. In the presence of VioE, the intermediate instead undergoes on-pathway [1,2] indole rearrangement to prodeoxyviolacein. The last two enzymes in the pathway are flavin-dependent oxygenases, VioC and VioD, that act sequentially. VioD hydroxylates one indole ring at the 5-position to yield proviolacein, and VioC then acts on the other indole ring at the 2-position to create the oxindole and complete violacein formation.     

Cloning of tryptophanase gene of Alcaligenes faecalis for effective production of l-tryptophan

For the effective production of l-tryptophan from indole and l-serine by the action of tryptophanase from Alcaligenes faecalis, cloning of the enzyme gene (tna) was studied. A. faecalis was transformed not only by broad host range plasmid pKT231 but also by pLG338, pACYC177, and pBR322 derivatives. A recombinant tna plasmid was isolated by shotgun experiments with Escherichia coli K-12, and the isolated tna gene located on the 3.2 kb DNA fragment. Tryptophanase activity of A. faecalis transformed by the tna recombinant plasmid was about 4-fold higher than that of wild cells.     

Synthesis and antimicrobial activity of β-carboline derivatives with N2-alkyl modifications

β-Carbolines constitute a vast group of indole alkaloids and exhibit various biological actions. The objective of this study was to investigate the structure–activity relationships of β-carboline derivatives on in vitro inhibitory effects against clinically relevant microorganisms. A series of β-carboline dimers and their N²-alkylated analogues were therefore prepared and evaluated for their antimicrobial effects. Among these, a dimeric 6-chlorocarboline N²-benzylated salt exerted potent activity against Staphylococcus aureus at MICs of 0.01–0.05?μmol/mL. Our work highlights that N¹-N¹ dimerization and N²-benzylation significantly enhanced the antimicrobial effects of compounds.     

Indoleamine 2,3-dioxygenase activity and l-tryptophan transport in human breast cancer cells

The activity and expression of indoleamine 2,3-dioxygenase together with l-tryptophan transport has been examined in cultured human breast cancer cells. MDA-MB-231 but not MCF-7 cells expressed mRNA for indoleamine 2,3-dioxygenase. Kynurenine production by MDA-MB-231 cells, which was taken as a measure of enzyme activity, was markedly stimulated by interferon-γ (1000 units/ml). Accordingly, l-tryptophan utilization by MDA-MB-231 cells was enhanced by interferon-γ. 1-Methyl-dl-tryptophan (1 mM) inhibited interferon-γ induced kynurenine production by MBA-MB-231 cells. Kynurenine production by MCF-7 cells remained at basal levels when cultured in the presence of interferon-γ. l-Tryptophan transport into MDA-MB-231 cells was via a Na⁺-independent, BCH-sensitive pathway. It appears that system L (LAT1/CD98) may be the only pathway for l-tryptophan transport into these cells. 1-Methyl-d,l-tryptophan trans-stimulated l-tryptophan efflux from MDA-MB-231 cells and thus appears to be a transported substrate of system L. The results suggest that system L plays an important role in providing indoleamine-2,3-dioxygenase with its main substrate, l-tryptophan, and suggest a mechanism by which estrogen receptor-negative breast cancer cells may evade the attention of the immune system.     

Levels of physiologically active indole derivatives in the fruiting bodies of some edible mushrooms (Basidiomycota) before and after thermal processing

Indole compounds were found in the fruiting bodies of selected mushroom species both before and after thermal processing. On the basis of HPLC analyses the following indole compounds were detected: l-tryptophan, 5-hydroxytryptophan, 5-methyltryptophan, tryptamine, 5-methyltryptamine, serotonin, indole and melatonin. The compound that was present in the largest amounts in the methanolic extracts from unprocessed mushrooms was 5-hydroxytryptophan in the amount of 7.32 mg/100 g DW in the case of the fruiting bodies of Auricularia polytricha; 15.83 in Suillus bovinus; 22.94 in Macrolepiota procera, and 24.83 in Lentinula edodes. In the methanolic extracts from thermally processed mushrooms the amount of 5-hydroxytryptophan was: 3.52 mg/100 g DW in the case of A. polytricha; 5.65 for Leccinum scabrum and 10.11 for M. procera. In addition, serotonin was found in unprocessed fruiting bodies of two mushroom species: 1.03 mg/100 g DW in L. edodes and 13.99 in L. scabrum, and also in thermally processed mushrooms.