【作者】 于珊珊；

【导师】 冯雁；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 来源于嗜热微生物的嗜热酶具有独特的生物稳定性和催化活性,对嗜热酶结构-功能关系的研究已得到越来越多科学工作者的关注,该领域研究不仅将加深酶学和酶工程理论的发展,也为酶的工业应用奠定了基础。论文第一部分克隆、表达、纯化和表征了来源于嗜热菌Fervidobacterium nodosum Rt17-B1的嗜热脂肪酶（FnL）和嗜热酯酶（FNE）。（1）FnL的序列同源性分析表明它与已知存在的脂肪酶基因的同源性低于30%,系统发育学分析表明它属于脂肪酶家族I,但与脂解酶家族I中现有的7个亚家族亲缘关系较远,因而将此嗜热脂肪酶所在的分支确定为脂肪酶家族I中的一个新的亚家族,并命名为family I.8;该重组脂肪酶的最适反应温度为70℃,最适pH为9.0,并且具有较高的热稳定性,其在60°C保温50小时几乎没有活力损失。底物选择性分析表明FnL偏好于水解中长链酰基(C₈-C₁₂)的p-nitrophenyl酯底物,最适底物为p-nitrophenyl caprate,另外,FnL还能够水解链长在C₄到C₁₆范围的三甘油酯底物,最适底物为三丁酸甘油酯（C₄）。结构建模和分子模拟研究揭示了FnL发挥催化作用的结构基础及其底物选择性的分子机制。（2）FNE的序列同源性分析表明其与已知存在的GDSL酯酶基因的同源性低于30%,在其序列中发现了SGNH亚家族蛋白的4个保守模块,即Block I,Block II,Block III和Block V,推测其可能的催化三联体为Ser⁸, Asp²¹⁵和His²¹⁸,氧洞基团为Ser⁸, Gly³³和Asn⁶⁶。该酯酶是GDSL家族中第一个来自嗜热微生物的酯酶。重组酶的最适反应温度为75℃,最适pH为8.5,FNE偏好于短链酰基（≤C₄）酯底物,最适底物为p-nitrophenyl acetate,它无法催化链长大于8的p-nitrophenyl酯底物的水解。通过与已知结构的酯酶进行结构比对,研究发现FNE位于活性位点之上存在一段插入序列,可能阻止了像三甘油酯等较大的底物分子与酶活性部位的接近,使FNE只能催化短链底物的水解。论文第二部分运用蛋白质晶体学的技术对重组FnL、FNE以及嗜热菌Geobacillus kaustophilus HTA426的磷酸三酯酶GK1506进行结晶和结构解析。成功地获得了磷酸三酯酶GK1506的结晶,使用分子置换法解析了分辨率达2.6 A的晶体结构。结构分析表明: GK1506蛋白的三维结构为TIM滚筒状结构,活性位点由β折叠的C末端以及连接这些折叠和螺旋之间的loop构成,同时活性部位结合有两个Co²⁺;另外GK1506蛋白结构具有与其他OPH蛋白相似的三个结合口袋,分别是大的结合口袋,小的结合口袋和离去集团结合口袋。另外我们还通过定点突变确定了参与催化作用的重要氨基酸残基为His23,His25,His178,His206,Lys145和Asp266。GK1506与有机磷底物的对接实验结果表明,在该酶活性中心上方存在一个由柔性较高的loop形成的盖子结构,推测该段loop所具有的“开-关”功能的结构变化可能与酶分子的底物进出和对有机磷底物或内酯类底物的选择性机制有关。以上关于嗜热磷酸三酯酶晶体结构的研究为揭示结构与功能关系以及为蛋白质合理设计方面的研究奠定了理论基础。更多还原

【Abstract】 The researches on thermophilic microorganisms and enzymes which have great impact on theoretical study and industrial applications have always been hot objects since 1990s’. This paper focuses on the characterization of the lipolytic hydrolyses from the thermophilic Fervidobacterium nodosum Rt17-B1 and the crystal structure of the phosphotriesterase from Geobacillus kaustophilus HTA426.In the first part of my thesis, a gene coding for a putative lipase composed of 302 amino acids from Fervidobacterium nodosum Rt17-B1 was cloned and characterized. Phylogenetic analysis suggests that this novel lipase represents a new subfamily of family I of bacterial lipases, annotated as family I.8. The recombinant protein can catalyze the hydrolysis of p-nitrophenyl esters and shows substrate preference for p-nitrophenyl caprate with a k_cat/K_m of 22500 s^-1μM^-1. Most importantly, it can hydrolyze triacylglycerols with long acyl chains. In the test conditions applied here it has a maximum activity at 70°C and pH 9.0 and displays extreme thermal stability. Interestingly, it was not only stable, but was also activated by treatment with polar organic solvents including propanol, acetone, dimethyl sulfoxide （DMSO） and N, N-dimethylformamide （DMF）. Structural modeling showed that it is composed of anα/β-hydrolase fold and a lid domain comprised of fourα-helices. A canonical catalytic triad consisting of Ser119, Asp206 and His282 was verified by site-directed mutagenesis. Moreover based on the constructed model, the substrate specificity was explained by analyses in the aspects of the binding free energy, spatial obstruction, and interactions between enzyme and substrate in the active site. Moreover, a putative bacterial esterase encoded by Fond₁301 with typical GDSL family motifs from Fervidobacterium nodosum Rt17-B1 was cloned and characterized. Recombinant FNE exhibited the highest esterase activity （14,000 U/mg） with p-nitrophenyl acetate （pNPC₂） as substrate. The catalytic efficiency (k_cat/K_m) toward p-nitrophenyl acetate （C2_） was approximately 120-fold higher than toward p-nitrophenyl butyrate （C₄）. No significant esterase activity was observed for the substrates with a chain length≥C₈. The monomeric enzyme has a molecular mass of 27.5 kDa and optimal activity around 75℃and at pH 8.5. Its thermostability is relatively high with a half-life of 80 min at 70℃, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the acetylesterase from Aspergillus aculeatus as a template. The model covered most of the FNE. The structure showed anα/β-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis. Sequence analysis showed that FNE is only distantly related to other esterases. A comparison of the conserved motifs shared with GDSL proteins revealed that FNE should be grouped into GDSL family and was further classified as SGNH hydrolase.In the second part of my thesis, the recombinant thermophilic phosphotriesterase GK1506 from Geobacillus kaustophilus HTA426 has been crystallized and the crystal structure of GK1506 has been determined by Singlewavelenth Anamolous Dispersion and molecular replacement.to 2.5 ?. The tertiary structure of this enzyme was anα/βbarrel in which the active site is located at the C-terminal ends of theβ-strands and in the loop regions connecting these strands to their subsequent helices. The active site of GK1506 contains the three substrate binding pockets found in other OPH enzymes: the small, large and leaving group pockets. The two metal ions was concluded in the active site of GK1506. site-directed mutagenesis lead us to make sure that His²³, His²⁵, His¹⁷⁸, His²⁰⁶, Lys¹⁴⁵ and Asp²⁶⁶ are important residues for catalytic function and substrate binding. The structure determination will illuminate the structure base of catalytic mechanisum. Moreover this knowledge can lead to the development of new and more efficient protein engineering strategies and a wide range of biotechnological applications.更多还原