Isotopes

同位素
  • 文章类型: Journal Article
    珊瑚礁的健康严重依赖珊瑚礁鱼类,然而过度捕捞导致了它们的下降,导致快速生长的藻类增加和珊瑚礁生态系统的变化,被描述为相移的现象。更清楚地了解食草之间的复杂相互作用,他们的食物,他们的肠道微生物组可以增强珊瑚礁的健康。这项研究检查了四种关键的名义上食草礁鱼种(Acanthuruschirurgus,Kyphosussp.,scarustrispinosus,和Sparisomaaxillare)在西南大西洋的Abrolhos礁系统中。这四种鱼类产生了大约1680万条16SrRNA序列,每个物种平均为317,047±57,007。细菌如变形杆菌,Firmicutes,蓝细菌在它们的微生物群中普遍存在。这些鱼类表现出独特的微生物群,这些微生物群是共同多样化的结果,饮食,限制移动。珊瑚相关细菌(内生单胞菌,根瘤菌,和Ruegeria)在鹦鹉鱼种Sc的肠道内容物中大量发现。trispinosus和Sp.axillare.这些鹦鹉鱼可以通过在珊瑚礁中传播这种有益的细菌来帮助珊瑚健康。同时,Kyphosussp.主要有Pirellulaceae和红杆菌科。四种鱼类的饮食由草皮成分(丝状蓝细菌)和褐藻(Dictyopteris)组成。它们也有相似的同位素生态位,暗示他们分享食物来源。观察到鱼类肌肉肠道组织的同位素特征和肠道内容物之间存在显着差异,指出宿主遗传学和肠道微生物在区分鱼类组织中的作用。
    Coral reefs rely heavily on reef fish for their health, yet overfishing has resulted in their decline, leading to an increase in fast-growing algae and changes in reef ecosystems, a phenomenon described as the phase-shift. A clearer understanding of the intricate interplay between herbivorous, their food, and their gut microbiomes could enhance reef health. This study examines the gut microbiome and isotopic markers (δ13C and δ15N) of four key nominally herbivorous reef fish species (Acanthurus chirurgus, Kyphosus sp., Scarus trispinosus, and Sparisoma axillare) in the Southwestern Atlantic\'s Abrolhos Reef systems. Approximately 16.8 million 16S rRNA sequences were produced for the four fish species, with an average of 317,047 ± 57,007 per species. Bacteria such as Proteobacteria, Firmicutes, and Cyanobacteria were prevalent in their microbiomes. These fish show unique microbiomes that result from co-diversification, diet, and restricted movement. Coral-associated bacteria (Endozoicomonas, Rhizobia, and Ruegeria) were found in abundance in the gut contents of the parrotfish species Sc. trispinosus and Sp. axillare. These parrotfishes could aid coral health by disseminating such beneficial bacteria across the reef. Meanwhile, Kyphosus sp. predominantly had Pirellulaceae and Rhodobacteraceae. Four fish species had a diet composed of turf components (filamentous Cyanobacteria) and brown algae (Dictyopteris). They also had similar isotopic niches, suggesting they shared food sources. A significant difference was observed between the isotopic signature of fish muscular gut tissue and gut contents, pointing to the role that host genetics and gut microbes play in differentiating fish tissues.
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  • 文章类型: Journal Article
    地下含水层中过量的致密非水相液体(DNAPLs)对人类健康和地下水资源的可持续发展构成了威胁。准确确定DNAPLs的来源对于地下水修复和预防工作至关重要。在以前的研究中,在使用同位素技术识别地下水中的DNAPLs方面取得了重大进展。在本文中,我们提供了用于源识别的常用同位素工具的全面概述。本概述将概述同位素技术的优点和局限性,并描述未来研究的需求。同位素追踪技术基于不同来源的DNAPLs的独特同位素特征,能够识别和区分DNAPL来源。δ13C和δ37Cl值最常用于识别地下水中的DNAPL。在现场应用中,然而,在经历了一系列人类和自然因素之后,来自不同来源的同位素特征的差异可以被削弱,这可能会影响源识别的准确性。为了提高DNAPL源识别的准确性,双同位素追踪方法似乎是最好的解决方案。尽管如此,面对复杂的污染环境,双同位素方法似乎捉襟见肘。因此,为了准确有效地评估地下水中DNAPLs的来源及其各自的贡献,还有待进一步的研究。这是地下水资源保护和修复工作的先决条件。
    Excessive dense nonaqueous phase liquids (DNAPLs) in subsurface aquifers posed a threat to human health and sustainable development of groundwater resources. Accurately identifying the sources of DNAPLs is crucial for groundwater remediation and prevention efforts. In the previous studies, significant advances were made in using isotope techniques for identifying DNAPLs in groundwater. In this paper, we provide a comprehensive overview of the commonly used isotopic tools applied to source identification. This overview will outline the advantages and limitations of the isotope technique and describe the needs for future research. Isotope tracing techniques are based on the unique isotopic characteristics of DNAPLs from different sources, enabling the identification and differentiation of DNAPL sources. The δ13C and δ37Cl values are most commonly used for identifying DNAPLs in groundwater. In field applications, however, the differences in isotopic characteristics from diverse sources can be weakened after undergoing a series of human and natural factors, which can affect the accuracy of source identification. To improve the accuracy of DNAPL source identification, a dual-isotope tracing approach seems the best available solution. Nonetheless, in the face of complex polluted environments, the dual-isotope method seems stretched. Therefore, further researches remain to be carried out to accurately and efficiently assess the sources of DNAPLs in groundwater and their individual contributions. This is a prerequisite for groundwater resource conservation and remediation efforts.
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  • 文章类型: Journal Article
    过量摄入碘对人体健康有害。近年来,高碘地下水已成为继全球关注的高砷、高氟地下水。深入了解影响地下水碘积累的环境因子和迁移转化机制,是有效防治地下水碘污染的科学前提。本文全面调查了地下水碘污染的相关文献,总结了富碘地下水的空间分布和水化学特征。系统总结了影响含水层碘富集的环境因素和水文地质条件。对水文地球化学的深入分析,物理化学,进行了表面环境中碘运输和转化的生物地球化学和人类影响,全面、系统地总结了高碘地下水研究领域的成果和结论。稳定同位素可作为追踪水化学成分来源的有力工具,生物地球化学过程,水文地质系统中地下水的补给源和流动路径,为高碘地下水系统的研究提供有效的研究方法和手段,加深对高碘地下水形成机制的认识,系统总结了同位素技术在高碘地下水中的应用,丰富了高碘地下水研究的方法和理论。本文为缺水地区地下水碘污染防治和地下水资源管理提供了较为科学的依据。
    Excessive intake of iodine will do harm to human health. In recent years, high iodine groundwater has become a global concern after high arsenic and high fluorine groundwater. A deep understanding of the environmental factors affecting iodine accumulation in groundwater and the mechanism of migration and transformation is the scientific prerequisite for effective prevention and control of iodine pollution in groundwater. The paper comprehensively investigated the relevant literature on iodine pollution of groundwater and summarized the present spatial distribution and hydrochemical characteristics of iodine-enriched groundwater. Environmental factors and hydrogeological conditions affecting iodine enrichment in aquifers are systematically summarized. An in-depth analysis of the hydrologic geochemistry, physical chemistry, biogeochemistry and human impacts of iodine transport and transformation in the surface environment was conducted, the results and conclusions in the field of high iodine groundwater research are summarized comprehensively and systematically. Stable isotope can be used as a powerful tool to track the sources of hydrochemical components, biogeochemistry processes, recharge sources and flow paths of groundwater in hydrogeological systems, to provide effective research methods and means for the study of high iodine groundwater system, and deepen the understanding of the formation mechanism of high iodine groundwater, the application of isotopic technique in high iodine groundwater is also systematically summarized, which enriches the method and theory of high iodine groundwater research. This paper provides more scientific basis for the prevention and control of groundwater iodine pollution and the management of groundwater resources in water-scarce areas.
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  • 文章类型: Journal Article
    大米消费是人类镉(Cd)暴露的主要途径。了解土壤-水稻系统中的Cd行为,特别是在野外条件下,是控制Cd积累的关键。这项研究分析了不同时间采样的水稻植物和表层土壤中的Cd浓度和同位素组成(δ114/110Cd),游县典型镉污染稻田居民的尿镉,湖南,中国。土壤水溶性Cd浓度随采样时间而变化,δ114/110Cdwater在排水条件下比洪水条件下轻,表明同位素较轻的Cd库补充水溶性Cd,增加Cd植物的可利用性。水溶性Cd和大气沉降均参与了水稻Cd的积累。由于土壤Cd植物利用率的增加,水溶性Cd的贡献从淹没条件下的28-52%增加到排水条件下的58-87%。大气沉积的贡献(12-72%)随着采样区之间潜在的大气沉积通量而增加。从根-茎-粒中富集重Cd同位素以防止水稻Cd积累。尿液中重同位素的富集程度不同表明Cd暴露源不同。这些发现为土壤-水稻系统中Cd的形态和植物有效性变化提供了有价值的见解,并强调了Cd同位素指纹在理解Cd的环境命运中的潜在应用。
    Rice consumption is a major pathway for human cadmium (Cd) exposure. Understanding Cd behavior in the soil-rice system, especially under field conditions, is pivotal for controlling Cd accumulation. This study analyzed Cd concentrations and isotope compositions (δ114/110Cd) in rice plants and surface soil sampled at different times, along with urinary Cd of residents from typical Cd-contaminated paddy fields in Youxian, Hunan, China. Soil water-soluble Cd concentrations varied across sampling times, with δ114/110Cdwater lighter under drained than flooded conditions, suggesting supplementation of water-soluble Cd by isotopically lighter Cd pools, increasing Cd phytoavailability. Both water-soluble Cd and atmospheric deposition contributed to rice Cd accumulation. Water-soluble Cd\'s contribution increased from 28-52% under flooded to 58-87% under drained conditions due to increased soil Cd phytoavailability. Atmospheric deposition\'s contribution (12-72%) increased with potential atmospheric deposition flux among sampling areas. The enrichment of heavy Cd isotopes occurred from root-stem-grain to prevent rice Cd accumulation. The different extent of enrichment of heavy isotopes in urine indicated different Cd exposure sources. These findings provide valuable insights into the speciation and phytoavailability changes of Cd in the soil-rice system and highlight the potential application of Cd isotopic fingerprinting in understanding the environmental fate of Cd.
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  • 文章类型: Journal Article
    同位素技术是追踪某些污染物来源或提供对环境过程的见解的理想工具。近年来,多收集器电感耦合等离子体质谱(MC-ICP-MS)的出现使各种金属稳定同位素的精确测量成为可能。由于各种环境样本中存在“指纹”属性,金属稳定同位素已被用于有效区分污染物的来源,并进一步了解相应的环境过程。金属元素的环境命运受到吸附的强烈控制,元素在溶解相和颗粒相之间分布的基本过程。金属元素在矿物和有机表面上的吸附显着影响其在环境中的生物地球化学循环。因此,阐明吸附过程中稳定金属同位素的分馏特性至关重要。在这次审查中,选择了三种典型的过渡金属元素,考虑Mo作为阴离子物种的代表,Fe和Zn作为阳离子物种的代表。对于Mo来说,较重的Mo同位素优先吸附在溶液相中,pH值对同位素分馏有更显著的影响,温度和离子强度相对不敏感。吸附过程中溶解和吸附的Mo之间的配位环境差异,即,附着模式(内球或外球)或分子对称性(例如,协调数和失真的幅度),可能是同位素分馏的原因。对于Fe,含水Fe(II)矿物中平衡/动力学Fe同位素分馏的研究并不简单。Fe(II)水溶液和Fe(羟基)氧化物之间的相互作用是复杂的和动态的。同位素效应是由于吸附的Fe(II)之间的电子和原子交换耦合,含水Fe(II),和活性Fe(III)在Fe(羟基)氧化物表面。对于Zn,较重的Fe同位素优先吸附在固相上,pH和离子强度是必不可少的影响因素。配位环境的差异可能是同位素分馏的原因。
    Isotope technology is an ideal tool for tracing the sources of certain pollutants or providing insights into environmental processes. In recent years, the advent of multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) has enabled the precise measurement of various metal stable isotopes. Due to the presence of \"fingerprint\" properties in various environmental samples, metal stable isotopes have been applied to distinguish the source of contaminants effectively and further understand the corresponding environmental processes. The environmental fate of metal elements is strongly controlled by adsorption, an essential process for the distribution of elements between the dissolved and particulate phases. The adsorption of metal elements on mineral and organic surfaces significantly affects their biogeochemical cycles in the environment. Therefore, it is crucial to elucidate the fractionation characteristics of stable metal isotopes during the adsorption process. In this review, three typical transitional metal elements were selected, considering Mo as the representative of anionic species and Fe and Zn as the representative of cationic species. For Mo, the heavier Mo isotope is preferentially adsorbed in the solution phase, pH has a more significant influence on isotope fractionation, and temperature and ionic strength are relatively insensitive. Differences in coordination environments between dissolved and adsorbed Mo during adsorption, i.e., attachment mode (inner- or outer-sphere) or molecular symmetry (e.g., coordination number and magnitude of distortion), are likely responsible for isotopic fractionation. For Fe, The study of equilibrium/kinetic Fe isotopic fractionation in aqueous Fe(II)-mineral is not simple. The interaction between aqueous Fe(II) and Fe (hydroxyl) oxides is complex and dynamic. The isotope effect is due to coupled electron and atom exchange between adsorbed Fe(II), aqueous Fe(II), and reactive Fe(III) on the surface of Fe (hydroxyl) oxide. For Zn, the heavier Fe isotope preferentially adsorbs on the solid phase, and pH and ionic strength are essential influencing factors. The difference in coordination environment may be the cause of isotope fractionation.
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  • 文章类型: Journal Article
    自上而下的质谱解卷积的目的是从原始质谱中的实验包络中识别单同位素峰。因此,准确评估理论和实验包络之间的相似性是质谱数据反卷积的关键步骤。现有的评估方法主要依赖于强度差异和m/z相似性,可能缺乏全面的评估。为了克服这种限制,并促进对理论和实验包络之间的相似性进行全面和精细的评估,必须系统地探索和确定评估这种对应关系的日益有效的特征。我们提出了用于同位素包络评估(FREE)的增强特征表示,该特征表示得出了各种特征表示,封装信封的基本物理属性,包括峰值强度和包络形状。我们训练了FREE,并评估了其在卵巢肿瘤(OT)(人类OT细胞)数据集和斑马鱼(ZF)(成熟女性ZF的大脑)数据集上的表现。具体来说,比较最先进的方法,FREE在OT和ZF数据集的多个评估指标中展示了更高的性能,特别强调精确度,并且它显示了根据得分在排名靠前的信封中更多的正信封的准确预测。此外,在ZF的跨物种数据集中,FREE确定了更高数量的蛋白型光谱匹配(PrSM),与EnvCNN相比,计数从50,795增加到52,927,FREE与TopFD的合并也表现出了一个值得称赞的能力来辨别117,883碎片离子,从而超过了通过应用EnvCNN与TopFD识别的97,554个碎片离子。为了进一步验证FREE的性能,我们已经测试了10个包含来自ProteomeXchange的36个子数据集的跨物种自上而下的蛋白质组。结果表明,与TopFD+FREE反卷积后,在第一轮和第二轮实验中,TopPIC在这10个数据集中识别出更多的PrSM。这些发现强调了FREE方法在不同蛋白质组中的鲁棒性和泛化能力。
    The aim of deconvolution of top-down mass spectra is to recognize monoisotopic peaks from the experimental envelopes in raw mass spectra. So accurate assessment of similarity between theoretical and experimental envelopes is a critical step in mass spectra data deconvolution. Existing evaluation methods primarily rely on intensity differences and m/z similarity, potentially lacking a comprehensive assessment. To overcome this constraint and facilitate a comprehensive and refined assessment of the similarity between theoretical and experimental envelopes, there exists an imperative to systematically explore and identify increasingly efficacious features for assessing this correspondence. We present enhanced feature representation for isotopic envelope evaluation (FREE) that derives diverse feature representations, encapsulating fundamental physical attributes of envelopes, including peak intensity and envelope shape. We trained FREE and evaluated its performance on both the ovarian tumor (OT) (human OT cells) data set and zebrafish (ZF) (brain in mature female ZF) data set. Specifically, comparing the state-of-art method, FREE demonstrates higher performance in multiple evaluation metrics across both the OT and ZF data sets, with a particular emphasis on precision, and it demonstrates accurate predictions of a greater number of positive envelopes among the top-ranked envelopes based on their scores. Moreover, within a cross-species data set of ZF, FREE identified a higher number of proteoform-spectrum matches (PrSMs), increasing the count from 50,795 to 52,927 compared to EnvCNN, the amalgamation of FREE with TopFD also exhibits a commendable capacity to discern 117,883 fragment ions, thus surpassing the 97,554 fragment ions identified through the application of EnvCNN in conjunction with TopFD. To further validate the performance of FREE, we have tested 10 a cross-species top-down proteomes containing 36 subdata set from ProteomeXchange. The results reveal that, after deconvolution with TopFD + FREE, TopPIC identifies more PrSMs across these 10 data sets in both the first and second rounds of experiments. These findings underscore the robustness and generalization capabilities of the FREE approach in diverse proteomes.
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  • 文章类型: Journal Article
    抗生素,他们的转型产品,抗生素抗性基因在环境中的易位给人类带来了巨大的健康风险,动物,和生态系统,与“一个健康”概念保持一致。人工湿地具有处理农业废水的巨大潜力,但未得到充分利用。生活污水,或污水处理厂的污染废水,以消除抗生素为目标。然而,对分布的全面了解,持久性,抗生素在人工湿地内的消散过程仍未被探索。在这种情况下,我们概述了在自然丰度下稳定同位素分析在抗生素中的当前应用。我们探索先进的多稳定同位素方法的机会,同位素概念可以有效地应用于检查湿地中抗生素的命运。使用多元素稳定同位素研究湿地抗生素的概念框架的发展引入了一种新的范式,为湿地系统中抗生素自然衰减的识别和量化提供了更深入的见解。这种观点有可能激励广大市民,政府机构,以及更广泛的研究团体,强调利用稳定同位素分析来研究湿地系统中的抗生素和其他新兴微污染物。
    Antibiotics, their transformation products, and the translocation of antibiotic-resistant genes in the environment pose significant health risks to humans, animals, and ecosystems, aligning with the One Health concept. Constructed wetlands hold substantial yet underutilized potential for treating wastewater from agricultural, domestic sewage, or contaminated effluents from wastewater treatment plants, with the goal of eliminating antibiotics. However, the comprehensive understanding of the distribution, persistence, and dissipation processes of antibiotics within constructed wetlands remains largely unexplored. In this context, we provide an overview of the current application of stable isotope analysis at natural abundance to antibiotics. We explore the opportunities of an advanced multiple stable isotope approach, where isotope concepts could be effectively applied to examine the fate of antibiotics in wetlands. The development of a conceptual framework to study antibiotics in wetlands using multi-element stable isotopes introduces a new paradigm, offering enhanced insights into the identification and quantification of natural attenuation of antibiotics within wetland systems. This perspective has the potential to inspire the general public, governmental bodies, and the broader research community, fostering an emphasis on the utilization of stable isotope analysis for studying antibiotics and other emerging micropollutants in wetland systems.
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  • 文章类型: Journal Article
    氮是地球上生命的重要元素。它在地表(大气地壳)和地幔之间的循环对大气和气候具有深远的影响。然而,我们对地球氮的起源和演化的理解仍然不完整。这篇综述概述了当前对地球氮收支和不同储层同位素组成的理解,深层氮地球化学的实验室限制,以及我们通过板块俯冲和火山活动对地球氮的起源和深层氮循环的理解。地球可能在主要吸积阶段异质地获得了氮,最初从减少,反辉石-球粒陨石样冲击器,随后来自日益氧化的撞击器和极小CI-球粒陨石样材料。像地球表面,地幔和岩心也是重要的氮储层。这三个储层的氮丰度和同位素组成可能在主要吸积阶段已经基本建立,并且在之后被深层氮循环所改变。尽管在现代俯冲带中存在净氮进入地球地幔的现象。然而,据估计,地球早期大气可能含有1.4倍于当今大气氮(PAN),0.4PAN通过生物固氮被隔离到地壳中。为了更好地了解地球氮的起源和演化,提出了未来研究的方向。
    Nitrogen is a vital element for life on Earth. Its cycling between the surface (atmosphere + crust) and the mantle has a profound influence on the atmosphere and climate. However, our understanding of the origin and evolution of Earth\'s nitrogen is still incomplete. This review presents an overview of the current understanding of Earth\'s nitrogen budget and the isotope composition of different reservoirs, laboratory constraints on deep nitrogen geochemistry, and our understanding of the origin of Earth\'s nitrogen and the deep nitrogen cycle through plate subduction and volcanism. The Earth may have acquired its nitrogen heterogeneously during the main accretion phase, initially from reduced, enstatite-chondrite-like impactors, and subsequently from increasingly oxidized impactors and minimal CI-chondrite-like materials. Like Earth\'s surface, the mantle and core are also significant nitrogen reservoirs. The nitrogen abundance and isotope composition of these three reservoirs may have been fundamentally established during the main accretion phase and have been insignificantly modified afterwards by the deep nitrogen cycle, although there is a net nitrogen ingassing into Earth\'s mantle in modern subduction zones. However, it is estimated that the early atmosphere of Earth may have contained ∼1.4 times the present-day atmospheric nitrogen (PAN), with ∼0.4 PAN being sequestered into the crust via biotic nitrogen fixation. In order to gain a better understanding of the origin and evolution of Earth\'s nitrogen, directions for future research are suggested.
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  • 文章类型: Journal Article
    尽管自然衰减是铀(U)污染的经济修复策略,有机分子在开采后含水层中驱动U自然衰减的作用尚不清楚。对地下水进行采样以调查化学物质,同位素,在通过中性原位浸出开采的典型U型矿床(钱家店U型矿床)中,生产井和开采后井的溶解有机质(DOM)成分及其与U自然衰减的关系。结果表明,Fe(II)浓度和δ34SSO4和δ18OSO4值增加,但是从生产井到开采后的井,铀浓度显着下降,表明Fe(III)还原和硫酸盐还原是导致U自然衰减的主要过程。微生物腐殖质样和蛋白质样成分介导Fe(III)和硫酸盐的还原,分别。H/C>1.5的有机分子有利于微生物介导的Fe(III)和硫酸盐的还原,并促进了溶解U的自然衰减。平均U衰减率为-1.07mg/L/yr,在大约11.2年内,受铀污染的地下水将自然减弱。该研究强调了特定的有机分子通过还原Fe(III)和硫酸盐来调节地下水U的自然衰减。
    Although natural attenuation is an economic remediation strategy for uranium (U) contamination, the role of organic molecules in driving U natural attenuation in postmining aquifers is not well-understood. Groundwaters were sampled to investigate the chemical, isotopic, and dissolved organic matter (DOM) compositions and their relationships to U natural attenuation from production wells and postmining wells in a typical U deposit (the Qianjiadian U deposit) mined by neutral in situ leaching. Results showed that Fe(II) concentrations and δ34SSO4 and δ18OSO4 values increased, but U concentrations decreased significantly from production wells to postmining wells, indicating that Fe(III) reduction and sulfate reduction were the predominant processes contributing to U natural attenuation. Microbial humic-like and protein-like components mediated the reduction of Fe(III) and sulfate, respectively. Organic molecules with H/C > 1.5 were conducive to microbe-mediated reduction of Fe(III) and sulfate and facilitated the natural attenuation of dissolved U. The average U attenuation rate was -1.07 mg/L/yr, with which the U-contaminated groundwater would be naturally attenuated in approximately 11.2 years. The study highlights the specific organic molecules regulating the natural attenuation of groundwater U via the reduction of Fe(III) and sulfate.
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  • 文章类型: Journal Article
    从烟曲霉中鉴定出二萜合酶AfAS。其氨基酸序列和-根据结构模型-活性位点结构与fusicocca-2,10(14)-二烯合酶PaFS高度相似,但是AfAS产生了结构更复杂的二萜,具有新型的6-5-5-5四环骨架,称为asperfumene。通过同位素标记实验和DFT计算阐明了AfAS的环化机理。反应级联在其初始步骤中通过与PaFS级联类似的中间体进行,但随后通过不寻常的邻位去质子化-再质子化过程发散,该过程在通往独特的asperfumene骨架的台阶入口处引发骨骼重排。结构模型仅揭示了活性位点之间的一个主要差异:在AfAS中,PaFS残基F65被I65取代。有趣的是,使用两种二萜合酶进行的定点诱变实验表明,位置65可作为生物合成四环反香烯与结构上较不复杂的二萜的双向功能开关。
    The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and-according to a structural model-active site architecture are highly similar to those of the fusicocca-2,10(14)-diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6-5-5-5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation-reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site-directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes.
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