Journal of structural biology最新文献_第6页

Structural insights into human adenylyl cyclase 9 in complex with Gαs by cryo-EM 人腺苷酸环化酶9与Gαs复合物的结构分析

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-06-02 DOI: 10.1016/j.jsb.2025.108223

Risa Nomura , Shota Suzuki , Koki Nishikawa , Hiroshi Suzuki , Yoshinori Fujiyoshi

Adenylyl cyclase 9 (AC9) regulates many physiologic functions through the production of cAMP, an important second messenger that regulates downstream effectors. The activation of AC9 is highly regulated by GPCR signaling. For example, AC9 is activated by the binding of Gαs, which, in turn, is activated by Gs-driven GPCRs. The structure of bovine AC9 (bAC9) was reported in 2019 using single-particle cryo-electron microscopy (cryo-EM). The structure of human AC9 (hAC9), however, has not been reported to date despite its potential benefit for drug development. Here, we analyzed the structures of hAC9 and hAC9 in complex with Gαs (hAC9-Gαs) using single-particle cryo-EM. The soluble domain of AC9-Gαs, the transmembrane (TM) domain of AC9-Gαs, and AC9 alone were analyzed at resolutions of 2.7 Å, 3.4 Å, and 3.2 Å, respectively. The results revealed three key aspects of the activation mechanism of hAC9 and its cAMP-generating function. First, a conformational change of the soluble domain was observed upon Gαs binding, resulting in a widely open catalytic site. Second, we analyzed the exact position of the C-terminus occluding the catalytic site in the hAC9-Gαs complex. Finally, we unexpectedly identified an elongated density suggestive of a single acyl chain in the TM domain. Consistent with recent reports on the allosteric regulation of AC by lipids, this finding suggests that the TM domain could serve as a potential drug target. These structural findings enhance our understanding of the structure and function of AC9 and other ACs and will provide a foundation for future AC-target drug discovery.

腺苷酸环化酶9 （AC9）通过产生cAMP来调节许多生理功能，cAMP是调节下游效应物的重要第二信使。AC9的激活受GPCR信号的高度调控。例如，AC9被g - αs结合激活，而g - αs又被gs驱动的gpcr激活。2019年，利用单粒子冷冻电镜（cryo-EM）报道了牛AC9 （bAC9）的结构。然而，人类AC9 （hAC9）的结构迄今尚未报道，尽管它对药物开发有潜在的益处。本文采用单粒子冷冻电镜分析了hAC9和hAC9与Gαs配合物（hAC9-Gαs）的结构。AC9- g - αs的可溶性结构域、AC9- g - αs的跨膜结构域和单独的AC9分别以2.7 Å、3.4 Å和3.2 Å的分辨率进行分析。结果揭示了hAC9的激活机制及其camp生成功能的三个关键方面。首先，在g - αs结合时观察到可溶性结构域的构象变化，导致催化位点广泛开放。其次，我们分析了hac9 - g - αs络合物中c -末端封闭催化位点的确切位置。最后，我们意外地发现了一个细长的密度，表明在TM结构域中有一个单酰基链。与最近关于脂质对AC变构调节的报道一致，这一发现表明TM结构域可能是一个潜在的药物靶点。这些结构上的发现增强了我们对AC9和其他ac的结构和功能的理解，并将为未来ac靶向药物的发现提供基础。

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引用次数: 0

A generalist deep-learning volume segmentation tool for volume electron microscopy of biological samples 一个通用的深度学习体积分割工具，用于生物样品的体积电子显微镜。

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-29 DOI: 10.1016/j.jsb.2025.108214

Yuyao Huang , Nickhil Jadav , Georgia Rutter , Lech Szymanski , Mihnea Bostina , Duane P. Harland

We present the Volume Segmentation Tool (VST), a deep learning software tool that implements volumetric image segmentation in volume electron microscopy image stack data from a wide range of biological sample types. VST automates the handling of data preprocessing, data augmentation, and network building, as well as the configuration for model training, while adapting to the specific dataset. We have tried to make VST more accessible by designing it to operate entirely on local hardware and have provided a browser-based interface with additional features for visualizations of the networks and augmented datasets. VST can utilise contour map prediction to support instance segmentation on top of semantic segmentation. Through examples from various resin-embedded sample derived transmission electron microscopy and scanning electron microscopy datasets, we demonstrate that VST achieves state of the art performance compared to existing approaches.

我们提出了体积分割工具（VST），这是一种深度学习软件工具，可在来自各种生物样品类型的体积电子显微镜图像堆栈数据中实现体积图像分割。VST自动处理数据预处理、数据增强和网络构建，以及模型训练的配置，同时适应特定的数据集。我们试图通过将VST设计为完全在本地硬件上运行来使其更易于访问，并提供了一个基于浏览器的界面，该界面具有用于网络可视化和增强数据集的附加功能。VST可以在语义分割的基础上利用等高线地图预测来支持实例分割。通过来自各种树脂包埋样品衍生的透射电子显微镜和扫描电子显微镜数据集的示例，我们证明与现有方法相比，VST实现了最先进的性能。

引用次数: 0

ArtiaX: geometric models, camera paths and image processing tools ArtiaX：几何模型，相机路径和图像处理工具。

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-28 DOI: 10.1016/j.jsb.2025.108215

Pauline Roth , Utz H. Ermel , Deborah Moser , Gunnar Arctaedius , Maren Wehrheim , Margot P. Scheffer , Achilleas S. Frangakis

Biomolecular image analysis and data interpretation is significantly improved through the application of advanced visualization techniques. Numerous visualization packages are currently available, spanning a broad spectrum of applications. Recently, we developed a plugin called ArtiaX which extended the capabilities of UCSF ChimeraX to address the specific demands of cryo-electron tomography. Here, we introduce the evolution of ArtiaX, that can now generate models to facilitate particle selection, define camera recording paths, and execute particle selection routines. Diverse models can be generated and populated with putative particle positions and orientations. In addition, models can be used to drive the camera position, thereby simplifying the process of movie creation. The plugin incorporates fundamental image filtering options for the on-the-fly analysis of tomographic data and provides compatibility of particle lists with RELION-5 .star files. Collectively, this update of ArtiaX comprehensively encompasses essential tools for the analysis and visualization of electron tomograms. It retains its hallmark attributes of speed, reliability, and user-friendliness, fostering seamless human–machine interaction.

通过应用先进的可视化技术，生物分子图像分析和数据解释得到了显著提高。目前有许多可视化包可用，涵盖了广泛的应用程序。最近，我们开发了一个名为ArtiaX的插件，扩展了UCSF ChimeraX的功能，以满足低温电子断层扫描的特定需求。在这里，我们介绍ArtiaX的演变，现在可以生成模型来促进粒子选择，定义相机记录路径，并执行粒子选择例程。不同的模型可以生成和填充假定的粒子位置和方向。此外，模型可以用来驱动摄像机的位置，从而简化了电影创作的过程。该插件包含基本的图像过滤选项，用于实时分析层析数据，并提供粒子列表与RELION-5的兼容性。明星的文件。总的来说，ArtiaX的更新全面包含了电子层析图分析和可视化的基本工具。它保留了其速度、可靠性和用户友好性的标志属性，促进了无缝的人机交互。

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引用次数: 0

Structural insights into substrate recognition of tri-modular xyloglucanase from Aspergillus oryzae 米曲霉三模木糖葡聚糖酶底物识别的结构研究。

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-23 DOI: 10.1016/j.jsb.2025.108213

Yusuke Nakamichi , Naoki Shimada , Masahiro Watanabe , Tatsuya Fujii , Katsuro Yaoi , Tomohiko Matsuzawa

Xeg5A from Aspergillus oryzae belongs to glycoside hydrolase family 5 subfamily 4. This enzyme has been characterized as a xyloglucan-specific endo-β-1,4-glucanase (xyloglucanase) that cleaves the main chain of xyloglucan at both unbranched and xylosylated glucosyl residues in an endo-processive mode of action. X-ray crystallography revealed that Xeg5A is a tri-modular enzyme composed of a catalytic, an Ig-like, and a C-terminal CBM46-like domains. Xeg5A structures complexed with branched xyloglucan oligosaccharides at subsites –4 to +4 showed that the recognition of xyloglucan side-chain moieties is important for Xeg5A activity. The crystal structure also provided structural insights into the role of the CBM46-like domain in contributing to regiospecificity and, possibly, processivity.

来自米曲霉的Xeg5A属于糖苷水解酶家族5亚家族4。该酶被认为是一种木葡聚糖特异性内切-β-1,4-葡聚糖酶（木葡聚糖酶），它以内切的方式在未支链和木基化的葡萄糖基残基上切割木葡聚糖的主链。x射线晶体学表明，Xeg5A是由催化结构域、类ig结构域和c端类cbm46结构域组成的三模块酶。Xeg5A结构在-4至+4亚位与支链木葡聚糖低聚糖络合，表明木葡聚糖侧链部分的识别对Xeg5A活性很重要。晶体结构还提供了对cbm46样结构域在促进区域特异性和可能的加工性方面的作用的结构见解。

引用次数: 0

Structural and biophysical characterization of PadR family protein Rv0047c of Mycobacterium tuberculosis H37Rv 结核分枝杆菌H37Rv PadR家族蛋白Rv0047c的结构与生物物理特性

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-20 DOI: 10.1016/j.jsb.2025.108211

Md Samsuddin Ansari , Muhammad Zohib , Meera Kumari , Vikash Yadav , Ravi Kant Pal , Sarita Tripathi , Anupam Jain , Bichitra Kumar Biswal , Ashish Arora

The members of the PadR family of transcriptional regulators are important for cell survival in toxic environments and play an important role in detoxification, pathogenicity, and multi-drug resistance. Rv0047c of Mycobacterium tuberculosis H37Rv is annotated as a PadR family protein. We have characterized the stability and structure of Rv0047c. Rv0047c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 55.3 °C. The crystal structure of Rv0047c was determined at a resolution of 3.15 Å. The structure indicates the biological unit to be a dimer with each monomer having a characteristic N-terminal winged-helix-turn-helix DNA binding domain and a C-terminal dimerization domain. The N-terminal domain is composed of four helices, α1, α2, α3, and α4 and two beta strands β1 and β2. The C-terminal dimerization domain (CTD) consists two long helices α6 and α7. The two domains are connected by helix α5. A short helical turn (helix αa, residue 89–92), leads to compaction of the α4-α5 loop. Rv0047c exhibits specificity in binding to an upstream region having an inverted repeat sequence. This binding is dependent upon Y18 and Y40 residue of Rv0047c, which are highly conserved among the PadR family. Overall, our results suggest a transcription regulatory role for Rv0047c, similar to other PadR family proteins.

PadR转录调控家族成员对毒性环境下的细胞存活至关重要，在解毒、致病性和多药耐药等方面发挥重要作用。分枝杆菌H37Rv的Rv0047c被注释为PadR家族蛋白。我们对Rv0047c的稳定性和结构进行了表征。Rv0047c在溶液中形成稳定的二聚体。其稳定性表征为热熔转变温度（Tm）为55.3 °C。Rv0047c的晶体结构以3.15 Å的分辨率确定。该结构表明该生物单元为二聚体，每个单体具有典型的n端翼-螺旋-旋-螺旋DNA结合结构域和c端二聚结构域。n端结构域由α1、α2、α3和α4四个螺旋和两条β链β1和β2组成。c端二聚化结构域（CTD）由两个长螺旋α6和α7组成。两个结构域由螺旋α5连接。短螺旋旋转（螺旋αa，残基89-92）导致α4-α5环的压实。Rv0047c在与具有反向重复序列的上游区域结合方面表现出特异性。这种结合依赖于Rv0047c的Y18和Y40残基，这些残基在PadR家族中高度保守。总的来说，我们的研究结果表明Rv0047c具有转录调控作用，类似于其他PadR家族蛋白。

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引用次数: 0

The mechanism underlying fascin-mediated bundling of actin filaments unveiled by cryo-electron tomography 冷冻电子断层扫描揭示了肌动蛋白纤维束化的机制

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-20 DOI: 10.1016/j.jsb.2025.108212

Xiyong Song , Jesús Baltanás-Copado , Muniyandi Selvaraj , Shrikant B. Kokate , Esa-Pekka Kumpula , Senena Corbalán-García , Juha T. Huiskonen

Fascins are crucial actin-binding proteins linked to carcinomas, such as cancer metastasis. Fascins crosslink unipolar actin filaments into linear and rigid parallel bundles, which play essential roles in the formation of filopodia, stereocilia and other membrane protrusions. However, the mechanism of how fascin bundles actin filaments has remained elusive. Here, we studied the organization of reconstituted fascin-actin bundles by cryo-electron tomography and determined the structure of the fascin–actin complex at 9 Å resolution by subtomogram averaging. Consistent with earlier findings, fascin molecules decorate adjacent actin filaments, positioned at regular intervals corresponding to the half-pitch of actin filaments. The fascin–actin complex structure allows us to verify the binding orientation of fascin between the two actin filaments. Fitting of the previously solved fascin crystal structure facilitates the analysis of the interaction surfaces. Our structural models serve as a blueprint to understand the detailed interactions between fascin and actins and provide new insights for the development of drugs targeting fascin proteins.

筋膜蛋白是与癌症相关的重要的肌动蛋白结合蛋白，如癌症转移。束状蛋白将单极肌动蛋白丝交联成线性和刚性平行束，在丝状足、立体纤毛和其他膜突起的形成中起重要作用。然而，束蛋白如何捆绑肌动蛋白细丝的机制仍然是难以捉摸的。在这里，我们通过冷冻电子断层扫描研究了重组的肌动蛋白束的组织，并通过亚断层扫描平均以9 Å分辨率确定了肌动蛋白复合物的结构。与早期的发现一致，束状蛋白分子装饰着相邻的肌动蛋白丝，其位置与肌动蛋白丝的半间距相对应。筋膜蛋白-肌动蛋白复合物结构使我们能够验证筋膜蛋白在两种肌动蛋白丝之间的结合方向。对先前解出的束状蛋白晶体结构进行拟合，便于对相互作用面的分析。我们的结构模型可以作为了解筋膜蛋白和肌动蛋白之间详细相互作用的蓝图，并为针对筋膜蛋白的药物开发提供新的见解。

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引用次数: 0

3D Multi-modal Imaging of demineralised dentine using combined synchrotron µ-XRD-CT and STXM-CT 同步加速器μ-XRD-CT与STXM-CT联合三维多模态成像牙本质脱矿。

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-16 DOI: 10.1016/j.jsb.2025.108208

Nathanael Leung , Robert A. Harper , Bin Zhu , Stuart A. Bartlett , Konstantin Ignatyev , Richard M. Shelton , Gabriel Landini , Tan Sui

Dental caries is the most prevalent oral disease that causes structural and compositional changes of the dental hard tissues due to a chronic demineralisation (combined with possible phases of remineralisation) process. Though considerable efforts have been directed at studying natural and artificial carious lesions, most characterisations remain either constrained to 2D analyses or have been unable to achieve fine resolution in 3D due to limited field of view. To overcome this challenge, the present study combined X-ray diffraction (XRD) and scanning transmission X-ray microscopy (STXM) tomography techniques to analyse the mineral density, scattering intensity, and crystallite size in normal, carious, 30 % artificially demineralised, and 50 % artificially demineralised dentine. Combined XRD and STXM tomography was performed on the I18 beamline at Diamond Light Source, using a 15 keV monochromatic beam with 2 × 2 µm spotsize and scanning with translation steps of 2 µm, providing a reconstructed voxel size of 2 × 2 × 2 µm. Natural carious dentine showed a reduction in hydroxyapatite (HAp) crystallite size due to chronic demineralisation. This was unlike artificially demineralised dentine samples that underwent short, continuous demineralisation, which created a zone of fully demineralised dentine, near the sample surface, and a zone of partially demineralised dentine that had a reduced mineral density but an increased average crystallite size.

龋齿是最常见的口腔疾病，由于慢性脱矿（结合可能的再矿化阶段）过程，导致牙齿硬组织的结构和成分变化。这些变化会影响最重要的口腔功能和美观，还会引起疼痛和不适。尽管在研究自然和人工龋齿病变方面已经做出了相当大的努力，但大多数特征仍然局限于2D分析，或者由于视野有限而无法在3D中实现精细分辨率。为了克服这一挑战，本研究结合x射线衍射（XRD）和扫描透射x射线显微镜（STXM）断层扫描技术，分析了正常、龋齿、30% %人工脱矿和50% %人工脱矿的牙本质中的矿物质密度、散射强度和晶体大小。采用15 keV单色光束，2 × 2 μm光斑尺寸，平移步长为2 μm，对金刚石光源下的I18光束线进行XRD和STXM联合层析成像，重建体素尺寸为2 × 2 × 2 μm。天然龋齿牙本质显示羟基磷灰石（HAp）晶体大小减少，由于慢性脱矿作用。这与人工脱矿的牙本质样品不同，人工脱矿的牙本质样品经历了短暂的、连续的脱矿，在样品表面附近形成了一个完全脱矿的牙本质区域，以及一个部分脱矿的牙本质区域，该区域的矿物密度降低，但平均晶粒尺寸增加。

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引用次数: 0

Calcium carbonate deposition in the spicules of the sponge Heteropia glomerosa (Porifera, Calcarea) 海绵小球异视（Porifera, Calcarea）的针状物中的碳酸钙沉积。

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-14 DOI: 10.1016/j.jsb.2025.108210

Camila Wendt , Fernanda C. de Medeiros , Raquel P. Gonçalves , Fabio Nudelman , Michelle Klautau , Marcos Farina , André L. Rossi

We investigated the biomineralization process of calcium carbonate deposition in the spicules of the calcareous sponge Heteropia glomerosa (Porifera, Calcarea). The finely polished spicules, composed of Mg-calcite, present a pattern of concentric lines spaced 400 nm apart when observed by scanning electron microscopy. We showed by electron backscattered diffraction that the whole spicule length has the same crystallographic orientation. Still, misorientation of up to 1.8° in adjacent regions (∼ 2 µm) and a continuous increase in the misalignment of up to 4.5° in regions separated by 300 µm were present. The sponge cells (mainly sclerocytes and pinacocytes) near the mineralization zone contain a high number of vesicles rich in calcium, which could be involved in the spicule biomineralization. We showed by electron and ion microscopies that the spicule growth occurs through the addition calcium carbonate granules, which form near the membrane of the sclerocyte, the cell responsible for biomineralization. The granules were deposited layer by layer on the surface of the spicule, increasing the biomineral thickness. Domains of 1–3 µm containing facets partially connected and surrounded by organic material were observed in an intermediate stage of the spicule growth. Misorientation between these domains was approximately 2°, similar to the misorientation obtained by electron backscattered diffraction, indicating that the spicule is formed by the addition of granules fusing in a predominant orientation.

我们研究了碳酸钙沉积在钙质海绵Heteropia glomerosa （Porifera, Calcarea）针状体中的生物矿化过程。由镁方解石组成的精细抛光针状体在扫描电镜下呈现出间距为400 nm的同心线模式。电子背散射衍射结果表明，整个针状体长度具有相同的晶体取向。尽管如此，在相邻区域（~ 2µm）存在高达1.8°的取向偏差，并且在间隔300 µm的区域存在高达4.5°的连续增加的取向偏差。矿化带附近的海绵细胞（主要是硬化细胞和松状细胞）含有大量富含钙的囊泡，这些囊泡可能参与了针状生物矿化。我们通过电子和离子显微镜显示，针状生长是通过添加碳酸钙颗粒发生的，碳酸钙颗粒在硬细胞膜附近形成，硬细胞负责生物矿化。颗粒一层一层地沉积在针状体表面，增加了生物矿物的厚度。在针状物生长的中间阶段，观察到1-3 µm的区域，其中包含部分连接并被有机物质包围的切面。这些畴之间的取向偏差约为2°，与电子背散射衍射得到的取向偏差相似，表明颗粒是由在优势取向上融合的颗粒添加而形成的。

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引用次数: 0

AITom: AI-guided cryo-electron tomography image analyses toolkit ai引导的低温电子断层成像图像分析工具包

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-14 DOI: 10.1016/j.jsb.2025.108207

Xueying Zhan , Xiangrui Zeng , Mostofa Rafid Uddin, Min Xu

Cryo-electron tomography (cryo-ET) is an essential tool in structural biology, uniquely capable of visualizing three-dimensional macromolecular complexes within their native cellular environments, thereby providing profound molecular-level insights. Despite its significant promise, cryo-ET faces persistent challenges in the systematic localization, identification, segmentation, and structural recovery of three-dimensional subcellular components, necessitating the development of efficient and accurate large-scale image analysis methods. In response to these complexities, this paper introduces AITom, an open-source artificial intelligence platform tailored for cryo-ET researchers. AITom integrates a comprehensive suite of public and proprietary algorithms, supporting both traditional template-based and template-free approaches, alongside state-of-the-art deep learning methodologies for cryo-ET data analysis. By incorporating diverse computational strategies, AITom enables researchers to more effectively tackle the complexities inherent in cryo-ET, facilitating precise analysis and interpretation of complex biological structures. Furthermore, AITom provides extensive tutorials for each analysis module, offering valuable guidance to users in utilizing its comprehensive functionalities.

低温电子断层扫描（cryo-ET）是结构生物学中必不可少的工具，具有独特的在其原生细胞环境中可视化三维大分子复合物的能力，从而提供深刻的分子水平见解。尽管前景广阔，但冷冻电镜在三维亚细胞成分的系统定位、识别、分割和结构恢复方面面临着持续的挑战，需要开发高效、准确的大规模图像分析方法。针对这些复杂性，本文介绍了为低温et研究人员量身定制的开源人工智能平台AITom。AITom集成了一套全面的公共和专有算法，支持传统的基于模板和无模板的方法，以及用于低温低温数据分析的最先进的深度学习方法。通过结合多种计算策略，AITom使研究人员能够更有效地解决cryo-ET固有的复杂性，促进复杂生物结构的精确分析和解释。此外，AITom还为每个分析模块提供了广泛的教程，为用户利用其综合功能提供了有价值的指导。

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引用次数: 0

Disulfide bonds enhance thermal stability and thumb region drives activity of the glycoside hydrolase 11 xylanase rMxylcd 二硫键增强热稳定性，拇指区驱动糖苷水解酶11木聚糖酶rMxylcd的活性。

IF 3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-05-12 DOI: 10.1016/j.jsb.2025.108209

Songna Wu , Nianying Zhang , Qun Wan

Thermostable enzymes have significant advantages in industries, yet uncovering novel candidates with superior properties remains a scientific pursuit. This study identified rMxyl^cd, a glycoside hydrolase 11 family thermophilic xylanase from compost-soil metagenome, which exhibited a high specific activity of 5954 U·mg⁻¹ at pH 5.5 and 80°C. rMxyl^cd was crystallized and diffracted to 1.5 Å resolution. Compared to the mesophilic xylanase Xyn II, rMxyl^cd exhibits a more compact architecture. Notably, B-factor analysis reveals a uniquely flexible thumb region, hinting at its critical role in the enzyme’s catalytic mechanism. rMxyl^cd contains two disulfide bonds in the thumb and the N-terminal regions. Breaking these disulfide bonds by mutagenesis has dramatically decreased activities and thermostability. Conversely, introducing an extra disulfide bond at the N-terminal region of its α-helix extended its half-life for more than five folds at 80°C. Our studies firmly establish that the disulfide bonds are essential for its high thermal stability and the flexibility of the thumb region is crucial for its activity. Comparing the rMxyl^cd crystal structure with the AlphaFold2-predicted model shows overall similarity, but the crystal structure offers higher local accuracy, especially in key functional regions. These findings not only deepen our understanding of the structure-function relationship of thermophilic xylanases but also inform a rational design of industrial enzymes.

耐热酶在工业上具有显著的优势，但发现具有优越性能的新型候选酶仍然是一项科学追求。本研究从堆肥-土壤元基因组中鉴定出糖苷水解酶11家族嗜热木聚糖酶rMxylcd，该酶在pH 5.5和80°C条件下的比活性为5954 U·mg-1。rMxylcd结晶，衍射至1.5 Å分辨率。与中温木聚糖酶Xyn II相比，rMxylcd具有更紧凑的结构。值得注意的是，b因子分析揭示了一个独特的灵活的拇指区域，暗示其在酶的催化机制中的关键作用。rMxylcd在拇指区和n端区含有两个二硫键。通过诱变破坏这些二硫键大大降低了活性和热稳定性。相反，在α-螺旋的n端区域引入一个额外的二硫键，在80℃时将其半衰期延长了5倍以上。我们的研究坚定地确立了二硫键对其高热稳定性和拇指区域的灵活性是至关重要的。将rMxylcd晶体结构与alphafold2预测模型进行比较，总体上相似，但晶体结构具有更高的局部精度，特别是在关键功能区域。这些发现不仅加深了我们对嗜热木聚糖酶结构-功能关系的认识，而且为工业酶的合理设计提供了依据。

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引用次数: 0