長期以來尿酸氧化酶被認為可以在銅離子存在的情況下催化尿酸降解成尿囊素的過程。之后當N’colloc’h和同事們第一次解析了黃曲霉菌中的尿酸氧化酶在2.05A分辨率的晶體結構后,人們發(fā)現(xiàn)尿酸氧化酶行使功能并不需要其它的輔因子。事實上,由于進化的原因,尿酸氧化酶在人類中并不存在,但由于它可以用來在人體內治療痛風,因此尿酸氧化酶獲得了許多科研和醫(yī)療上的注意。痛風是一種嚴重的關節(jié)疾病,會導致關節(jié)或關節(jié)腔的腫痛,幾乎對每個種族的成年婦女造成影響。而尿酸的水平的異常提高則是痛風的重要標志。目前可以通過直接在人體內注射尿酸氧化酶來抑制尿酸水平的提高。研究人員認為尿酸氧化酶是一個治療高尿酸和痛風的理想藥物。目前通過在酵母中表達黃曲霉菌中的尿酸氧化酶,該酶的藥物制劑（商業(yè)用名Fasturtec）已被用于上述病癥的臨床治療,但它在一些人群中會引發(fā)高鐵血紅蛋白血癥的嚴重副作用。目前已報道的結構顯示,尿酸氧化酶具備一個同四聚體結構,該結構被認為對于它的酶活和結構穩(wěn)定性非常重要。它的底物結合口袋已獲得大量研究,但仍有一些細節(jié)有待進一步的揭示。我們在本研究中報道枯草桿菌168中尿酸氧化酶2.6A的晶體結構。通過和... 

【文章來源】：中國科學技術大學安徽省 211工程院校 985工程院校

【文章頁數(shù)】：99 頁

【學位級別】：博士

【文章目錄】：
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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