長(zhǎng)期以來(lái)尿酸氧化酶被認(rèn)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(guò)程。之后當(dāng)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(jié)構(gòu)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(shí)上,由于進(jìn)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(lái)在人體內(nèi)治療痛風(fēng),因此尿酸氧化酶獲得了許多科研和醫(yī)療上的注意。痛風(fēng)是一種嚴(yán)重的關(guān)節(jié)疾病,會(huì)導(dǎo)致關(guān)節(jié)或關(guān)節(jié)腔的腫痛,幾乎對(duì)每個(gè)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(fēng)的重要標(biāo)志。目前可以通過(guò)直接在人體內(nèi)注射尿酸氧化酶來(lái)抑制尿酸水平的提高。研究人員認(rèn)為尿酸氧化酶是一個(gè)治療高尿酸和痛風(fēng)的理想藥物。目前通過(guò)在酵母中表達(dá)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(huì)引發(fā)高鐵血紅蛋白血癥的嚴(yán)重副作用。目前已報(bào)道的結(jié)構(gòu)顯示,尿酸氧化酶具備一個(gè)同四聚體結(jié)構(gòu),該結(jié)構(gòu)被認(rèn)為對(duì)于它的酶活和結(jié)構(gòu)穩(wěn)定性非常重要。它的底物結(jié)合口袋已獲得大量研究,但仍有一些細(xì)節(jié)有待進(jìn)一步的揭示。我們?cè)诒狙芯恐袌?bào)道枯草桿菌168中尿酸氧化酶2.6A的晶體結(jié)構(gòu)。通過(guò)和... 

【文章來(lái)源】：中國(guó)科學(xué)技術(shù)大學(xué)安徽省 211工程院校 985工程院校

【文章頁(yè)數(shù)】：99 頁(yè)

【學(xué)位級(jí)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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