Characterization of a Tryptophan 6-Halogenase from Streptomyces albus and Its Regioselectivity Determinants

Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l -tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.     

The structural consequences of exchanging tryptophan and tyrosine residues in B.stearothermophilus lactate dehydrogenase

A mutant Bacillus stearothermophilus lactate dehydrogenase hasbeen prepared in which all three tryptophan residues in thewild-type enzyme have been replaced by tyrosines. In addition,a tyrosine residue has been mutated to a tryptophan, which actsas a fluorescence probe to monitor protein folding. The mutantenzyme crystallizes in the same crystal form as the wild-type.The crystal structure of the mutant has been determined at 2.8Å resolution. Solution studies have suggested that thereis little effect upon the mutant enzyme as judged by its kineticproperties. Comparison of the crystal structures of the mutantand wild-type enzymes confirms this conclusion, and revealsthat alterations in structure in the region of these mutationsare of a similar magnitude to those observed throughout thestructure, and are not significant when compared with the errorsin atomic positions expected for a structure at this resolution.     

酶法合成中色氨酸的测定

建立了光度测定酶法合成中色氨酸含量的新体系。该检测体系不仅能准确地测定产物色氨酸的含量，且对底物吲哚能同时测定。有效地避免了其他色氨酸测定方法底物吲哚的干扰，通过标准加入法色氨酸及吲哚的回收率分别为９７．８％～１０３．３％，９７．６％～１０３％。实验证明该体系简单、实用，测定结果可靠。     

Mixture Effects of Tryptophan Intestinal Microbial Metabolites on Aryl Hydrocarbon Receptor Activity

Aryl hydrocarbon receptor (AHR) plays pivotal roles in intestinal physiology and pathophysiology. Intestinal AHR is activated by numerous dietary, endogenous, and microbial ligands. Whereas the effects of individual compounds on AHR are mostly known, the effects of real physiological mixtures occurring in the intestine have not been studied. Using reporter gene assays and RT-PCR, we evaluated the combinatorial effects (3520 combinations) of 11 microbial catabolites of tryptophan (MICTs) on AHR. We robustly (n = 30) determined the potencies and relative efficacies of single MICTs. Synergistic effects of MICT binary mixtures were observed between low- or medium-efficacy agonists, in particular for combinations of indole-3-propionate and indole-3-lactate. Combinations comprising highly efficacious agonists such as indole-3-pyruvate displayed rather antagonist effects, caused by saturation of the assay response. These synergistic effects were confirmed by RT-PCR as CYP1A1 mRNA expression. We also tested mimic multicomponent and binary mixtures of MICTs, prepared based on the metabolomic analyses of human feces and colonoscopy aspirates, respectively. In this case, AHR responsiveness did not correlate with type of diet or health status, and the indole concentrations in the mixtures were determinative of gross AHR activity. Future systematic research on the synergistic activation of AHR by microbial metabolites and other ligands is needed.     

聚色氨酸修饰玻碳电极测定动物性食品中己烯雌酚

用循环伏安法制备了聚色氨酸修饰玻碳电极。研究了己烯雌酚在聚色氨酸修饰电极上的电化学行为,建立了一种直接测定动物性食品中己烯雌酚含量的新方法。实验结果表明:在p H=5.0的磷酸盐缓冲溶液体系中,己烯雌酚在聚色氨酸修饰玻碳电极上于-0.2⁰.8 V范围内出现一对可逆的氧化还原峰,与裸玻碳电极相比较,聚色氨酸修饰电极对己烯雌酚的电化学反应具有促进作用。己烯雌酚浓度在5.00×10^-81.00×10^-6mol/L范围内与其氧化峰电流成正比,检出限为2.00×10^-9mol/L,对己烯雌酚溶液平行测定6次的RSD为2.4%。该修饰电极具有良好的灵敏度、选择性和稳定性,可用于牛肉、鱼肉及奶粉中己烯雌酚含量的测定,回收率为96.0%¹02.3%,结果满意,方法具有实用价值。      

LC-MS/MS法测定小鼠脑组织中色氨酸及其3种代谢物

目的:建立检测小鼠脑组织中色氨酸、5-羟基色氨酸、5-羟色胺和5-羟基吲哚乙酸含量的LC-MS/MS分析方法。方法:采用TSK Gel amide 80（2.0 mm×15 cm,3μm）色谱柱,柱温35℃,乙腈:甲酸铵水溶液（15 mmol·L-1,p H5.5）（40∶60,v/v）为流动相,流速0.4 m L·min-1,采用正离子多反应监测（MRM）模式进行扫描。结果:4种待测物均在5 min内完成测定,色氨酸、5-羟基色氨酸、5-羟色胺和5-羟基吲哚乙酸最低定量限分别为0.2、0.1、0.2、0.2 ng·m L-1,最低检测限分别为0.05、0.02、0.05、0.05 ng·m L-1,在1⁵00 ng·m L-1浓度范围内线性关系均良好,方法的精密度和加样回收率均符合生物样品的分析要求。将该方法应用于口服萱草花总黄酮的小鼠脑提取物检测后发现:脑内四种待测物浓度明显发生改变,且变化规律与阳性药物帕罗西汀相同。结论:本方法简便、准确、灵敏度高,专属性强,重复性好,适用于脑组织中色氨酸-五羟色胺代谢过程中的4种成分的测定,可应用于神经精神类药物的药理作用机制研究。      

色氨酸残基在双功能酶CCBE中的作用

应用化学修饰剂NBS对从绿色木霉生产的纤维素酶中分离出的既具有壳聚糖酶活性又具有纤维素酶活性的单一组分(简称双功能酶CCBE)进行了修饰。结果表明,CCBE的壳聚酶活性中心需一分子色氨酸残基,CMCase活性中心需两分子色氨酸残基;底物保护作用及酶解动力学研究表明,CCBE的壳聚酶活性中心及CMCase活性中心的一分子的色氨酸残基位于底物结合部位,而CMCase活性中另一分子色氨酸残基位于催化结构中心。     

Administration of xylo-oligosaccharides improves depressive-like behaviour in mice caused by chronic unpredictable mild stress by altering microbiota composition

Xylo-oligosaccharides (XOS) is a functional oligosaccharide with prebiotic properties, and could be fermented by gut microbiota. In this study, the modulatory effects of XOS on emotions and behaviours at different dose were investigated based on the mouse model of chronic unpredictable mild stress-induced depression. The result showed that 0.23 and 0.50 g kg⁻¹ doses of XOS significantly increased sucrose preference index and decreased the immobility time in tail suspension and forced swimming tests. Meanwhile, the dose of 0.23 g kg⁻¹ day⁻¹ XOS significantly increased the concentration of neurotransmitters, such as 5-hydroxytryptamine and γ- aminobutyric acid. Furthermore, the analysis of the faecal microbiota with XOS intervention showed a higher relative abundance of Akkermansia, Bifidobacterium and Lactobacillus, fewer Desulfovibrio and the SCFA concentrations increased with XOS prevention treatment. These results suggested that the dietary supplementation XOS could improve chronic stress-induced depressive-like behaviour by modulating neurotransmitters and reverting gut microbiota changes in mice. These findings present XOS as a potential prebiotic targeting to the gut microbiota for mood disorders.