枯草杆菌尿酸氧化酶晶体结构的测定与分析

发布时间：2021-02-08 09:55

　　长期以来尿酸氧化酶被认为可以在铜离子存在的情况下催化尿酸降解成尿囊素的过程。之后当N’colloc’h和同事们第一次解析了黄曲霉菌中的尿酸氧化酶在2.05A分辨率的晶体结构后,人们发现尿酸氧化酶行使功能并不需要其它的辅因子。事实上,由于进化的原因,尿酸氧化酶在人类中并不存在,但由于它可以用来在人体内治疗痛风,因此尿酸氧化酶获得了许多科研和医疗上的注意。痛风是一种严重的关节疾病,会导致关节或关节腔的肿痛,几乎对每个种族的成年妇女造成影响。而尿酸的水平的异常提高则是痛风的重要标志。目前可以通过直接在人体内注射尿酸氧化酶来抑制尿酸水平的提高。研究人员认为尿酸氧化酶是一个治疗高尿酸和痛风的理想药物。目前通过在酵母中表达黄曲霉菌中的尿酸氧化酶,该酶的药物制剂（商业用名Fasturtec）已被用于上述病症的临床治疗,但它在一些人群中会引发高铁血红蛋白血症的严重副作用。目前已报道的结构显示,尿酸氧化酶具备一个同四聚体结构,该结构被认为对于它的酶活和结构稳定性非常重要。它的底物结合口袋已获得大量研究,但仍有一些细节有待进一步的揭示。我们在本研究中报道枯草杆菌168中尿酸氧化酶2.6A的晶体结构。通过和... 

【文章来源】：中国科学技术大学安徽省 211工程院校 985工程院校

【文章页数】：99 页

【学位级别】：博士

【文章目录】：
DEDICATION
摘要
Abstract
Abbreviations
Chapter 1: Introduction
    1.1. Gout
    1.2. Gout and its etiology
        1.2.1. Genetic factors:
        1.2.2. Non Genetic Factors
    1.3. Clinical evaluation of Gout
        1.3.1. Hyperuricemia
        1.3.2. Signs and Symptoms
        1.3.3. Diagnostic test and Gout estimation
    1.4. Pathophysiology of Gout
        1.4.1. Mechanism and causes of Hyperuricemia
        1.4.2. Production and deposition of urate crystals
        1.4.3. Mechanisms of immune responses
        1.4.4. Stages of Inflammation in Gout
    1.5. Physiology of Hyperuricemia
        1.5.1. Uric acid production and metabolism
        1.5.2. Physiological functions of Uric acid
        1.5.3. Maintenance of Uric acid homeostasis
    1.6. Management of Gout
    1.7. Urate oxidase: an oxygenase
    1.8. Structure of an archetypical urate oxidase:
    1.9. Importance of urate oxidase
Chapter 2: Material and Methods
    2.1. Urate oxidase protein cloning construction
        2.1.1. Molecular cloning
        2.1.2. Purpose Restriction of DNA fragments
        2.1.3. Preparation of the vector:
        2.1.4. Ligation of the DNA fragment to the vector
        2.1.5. Transformation of the recombinant plasmid to the competent cells
    2.2. Protein Expression and Purification
        2.2.1. Expression of Bacillus subtilis urate oxidase
        2.2.2. Extraction and Purification:
        2.2.3. Crystallization, data collection and processing
    2.3. Small angle X-ray scattering measurements
    2.4. Urate oxidase activity assay
Chapter 3: Results
    3.1. Cloning, Expression and Purification of Bacillus subtilis urate oxidase
    3.2. Structure determination of Bacillus subtilis urate oxidase using X-ray Crystallography
    3.3. Analysis of Crystal structure of urate oxidase enzyme
    3.4. Structure Analysis using Small Angle X-ray scattering
    3.5. Assessment of the substrate binding pocket by superimposition of BsUOX with a urateoxidase-uric acid complex structure
    3.6. Sequence analysis and Comparison of BsUOX with urate oxidases from other bacterialspecies
    3.7. Structural analysis and Comparison of BsUOX with urate oxidases from other bacterialspecies
    3.8. BsUOX structure has an unusual C-terminal fold
    3.9. Protein cloning, expression and purification of BsUOX mutants
Chapter 4: Discussion
Chapter 5: References
ACKNOWLEDGMENTS
Academic papers Published during PhD period



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