【作者】 郑柏松；

【导师】 冯雁；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 来源于嗜热菌的酶类具有极好的高温反应活性和稳定性,在能源、化工、食品和医药等行业展现出越来越广阔的应用潜力。解析嗜热酶及酶-底物复合物的晶体结构,研究其结构特征,将有助于揭示酶超常生物学稳定性以及高温催化反应机制的分子基础,为发掘嗜热酶的新功能、指导酶分子设计和改造等工作提供必要的理论依据。论文第一部分运用分子生物学技术克隆、表达、纯化了来源于嗜热菌Fervidobacterium nodosum Rt17-B1的嗜热β-1,4-葡聚糖内切酶FnCel5A及其N末端截短突变体FnCel5AND,并运用蛋白质晶体学的技术进行结晶,最终使用单波长反常散射法解析了分辨率达1.7 ?的FnCel5A和2.2 ?的复合物FnCel5AND:Glucose的晶体结构。结构分析表明,该酶具有糖基水解酶第五家族典型的(β/α)8形式TIM桶状结构;与中温和低温结构同源物比较,FnCel5A在氨基酸组成和分子内相互作用等方面具备高稳定性酶的结构特征;确定了Glu283-His126和Glu167-His226双催化活性中心,以及底物结合残基;结合分子模拟,提出了FnCel5A与底物结合的模型,阐明了该酶水解直链糖底物的催化机理。论文第二部分克隆、表达、表征了来源于嗜热古菌的酰基肽水解酶N末端截短突变体ApAPH-ΔN21;结晶并利用分子置换法解析了2.5 ?分辨率的突变体结构。结构分析和性质测定结果显示,ApAPH-ΔN21较野生型的最适反应温度降低18℃,对热和化学变性剂抗性降低;平均结构B因子由野生型的19.4 ?2升高到突变体的28.4 ?2。以上证据表明保守的N末端的对于保持ApAPH或家族中其他成员的稳定性具有重要作用。更多还原

【Abstract】 Enzymes produced by thermophiles and hyperthermophiles are typically thermostable (resistant to irreversible inactivation at high temperatures) and thermophilic (i.e. optimally active at high temperatures) and are known as thermozymes. In view of these important advantages, thermozymes are attracting much industrial interest. The structure determination of thermostable enzymes and their complex with substrate or inhibitor, and investigation of the structural feature of stability will illuminate the structure base of stability and catalytic mechanisum at high temperature. This knowledge can lead to the development of new and more efficient protein engineering strategies and a wide range of biotechnological applications.Cellulose, the most abundant organic source of feed, fuel and chemicals consists of glucose units linked byβ-1,4-glycosidic bonds in a linear mode. The difference in the type of bond and the highly ordered crystalline form of the compound between starch and cellulose make cellulose more resistant to digest and hydrolyze. The enzymes required for the hydrolysis of cellulose include endoglucanases, exoglucanases andβ-glucosidases. While cellulase is an endoglucanase that hydrolyses cellulose randomly, producing oligosacaccharides, cellobiose and glucose, exoglucanases hydrolyzeβ-1,4-D-glucosidic linkages in cellulose releasing cellobiose from the non-reducing end. On the other hand,β-glycosidases of thermophilic origin, which have received renewed attention in the pharmaceutical industry hydrolyze cellobiose to glucose.In the current industrial processes, cellulolytic enzymes are employed in the color extractions of juices, in detergents causing color brightening and softening, in the biostoning of jeans, in the pretreatment of biomass that contains cellulose to improve nutritional quality of forage and in the pretreatment of industrial wastes. But cellulases, which we have already used, are still unsatisfactory. For instance, the biopolishing process of cotton in the textile industry requires cellulase stable at high temperature close to 100℃. Presently used enzymes for this purpose, however, are active only at 50–55℃. Cellulase production is also found to be the most expensive step during ethanol production from cellulosic biomass, and accounted for approximately 40% of the total cost. So, thermophilic cellulases with high activity and high stability are required to attack the native crystalline cellulose, which is water insoluble and occurs as fibers of densely packed structures.In our previous study, the thermophilic endo-β-1,4-glucanase FnCel5A was first cloned from Fervidobacterium nodosum Rt17-B1 and overexpressed in E. coli. The optimum temperature and pH of FnCel5A were 80℃and 5.5-5.8, respectively. The recombinant enzyme had a high endo-β-1,4-glucan activity. The enzyme was high thermal active with t1/2 was 48 h at 80℃, and can be thermal activated by 40% at 70℃、75℃、80℃for 2 h. The activity of FnCel5A was totally inhibited by Zn2+ (relative remain activity 3 %), and partially inhibited by Ni2+, Co2+and K+ (relative remain activity 73%, 79% and 88%). Structure determination of thermostable cellulases and their complex with substrate or inhibitor, and investigation of the structural feature of stability will illuminate the structure base of stability and catalytic mechanisum at high temperature. This knowledge not only can lead to find the new function of the enzyme, but also can direct molecular design and modification of present cellulases. It will accelerate the development of new and more efficient protein engineering strategies and start a wide range of biotechnological applications.In the first part of my thesis, the thermophilic endo-β-1,4-glucanase FnCel5A and the N-terminal truncated mutant FnCel5AND were cloned, overexpressed and purified from Fervidobacterium nodosum Rt17-B1. The recombinant has been crystallized and the crystal structure of FnCel5A and complex FnCel5AND:Glucose have been determined by Singlewavelenth Anamolous Dispersion and molecular replacement.to 1.7 ? and 2.5 ? respectively. Crystal structure shows that FnCel5A presents the (β/α)8-barrel structure typical of clan GH-5 of glycoside hydrolase families. Compare with the homologous from mesophiles and psychrophiles, it suggests that FnCel5A owns the structure feature of thermostable enzyme in amino acid composition and intermolecular interaction. Structure analysis of the complex FnCel5AND:Glucose lead us to make sure that Asn51, Trp61, His126, His127, Asn166, Glu167, Trp204, His226, Tyr228, Phe231, His235, Trp240, Glu283, Trp316 and Phe322 are important residues for catalytic function and substrate binding. Combined with the molecular simulations, the model of substrate binding was established. The model described the catalytic mechanisum of the straight chain substrate of glycosyl in detail. Termini of proteins are usually the regions with highest thermal factors in a protein structure. They are likely to unfold first during thermal denaturation. Many proteins whose stability are modified by the N-terminal structure and find that extra extension, deletion of the terminus and disruption of the interaction between the terminus and the structure will alter the stability of the protein. Researchers speculate that some protein termini are the original nucleus of protein folding and the high thermostability is the result of the tight binding of the terminus with the protein. In our previous report, the APH gene from the thermophilic archaeon Aeropyrum pernix K1 has been cloned and overexpressed in Escherichia coli. The enzyme possesses both acylpeptide hydrolase and esterase activities. It is extremely thermostable. We determined the crystal structure feature of ApAPH, which include anα/βhydrolase domain, and aβ-propeller domain. The N-terminus of the POP family provides the outermost strand, which extends from theβ-propeller domain and packs against theα/βhydrolase domain. Homology analysis by sequence and structure reveals that ApAPH has the shortest N-terminal extension, which raised an intriguing question about the function of the most conserved N-terminus on protein stability and catalysis. Moreover, analysis of the ApAPH structure reveals that N-terminus anchoring is achieved through ion pairing and hydrophobic interactions. Since the N-terminal helix of APH is conserved in all members of the POP family, we speculate that this region may play an important role in either function or stability, or both.To investigate the thermostable mechanism of N-terminus, ApAPH and N-terminal truncated mutant ApAPH-ΔN21 were cloned, overexpressed and purified in the second part of the thesis. The recombinant ApAPH-ΔN21 has been crystallized and the crystal structure has been determined by molecular replacement. The main-chain conformations of them are very similar, except for theα-helix at the N-terminus. The root mean square (r.m.s.) deviation of all the main-chain atoms between the two structures is 0.954 ?. It means that there is no significant change between two structures.The character of enzymatic activity and stability were determined by enzymatic assay, spectroscopy and structural analysis. Compare with the wild type, the thermostable mechanism of N-terminus of ApAPH was elucidated.(1) The N-terminal helix is important for the enzyme’s thermophilic function.Although apAPH shows high structure similarity with several members of the POP family, in particular, on their secondary structures, these enzymes have diverse N-terminal extensions. Previously, it has been suggested that a tight binding of the N-terminal region to the catalytic domain in hyperthermophiles is one of the mechanisms for their high thermostability. apAPH possesses the shortest N terminal extension among all known POP family members and the secondary structure element in this region is conserved among all members of the POP family. The purpose of this paper is to examine the role of the apAPH N-terminal extension in stability and enzymatic activity. As expected, deletion of the N-terminal region leads to the shift of the optimal temperature to a lower value by ~18°C, confirming that the N-terminal helix region is critical for the hyperthermophilic function of the enzyme. Since thermophilic archaea is at the bottom of the evolution tree, we anticipate that this terminal region may play an important role in all POP family members.(2) The salt bridges have minor effects on the stability of the enzyme.Analysis of apAPH crystal structure reveals that the two salt bridges Asp15-Arg355 and Arg18-Asp325 are involved in connecting the N-terminal extension to the catalytic domain at the surface of the protein. Further examination of the crystal structures of the members in the POP family reveals that the two salt bridges also exist in porcine POP numbering (R60-D695 and K64-E691). Based on our results, disruption of these highly conserved salt bridges has only minor effects on the conformation, stability, and catalytic properties of enzymes. The result illustrates that the lack of such salt bridges in the N terminal region of apAPH leads to only small changes in stability and catalytic function. In the case of mutant D15A/R18A, almost all the results about activity and stability of mutant D15A/R18A are closer to the wild type protein than other mutants. Molecular dynamic simulation reveals that two new salt bridges (Arg11-Asp325 and Arg14-Asp325) and more hydrophobic interactions are formed. Therefore the thermostability of the double mutant recovered. These results suggest that other factors such as hydrophobic interactions between the catalytic domain and the N-terminal region are likely to be responsible for the stabilization of the WT enzyme.(3) Deletion of N-helix increases the conformation flexibility and enzymatic activity at low temperature.The accumulation of crystallographic data on thermostable and hyperthermostable enzymes, together with statistic analyses permitted by the rapid advancement of genome sequencing projects, has substantially improved our understanding of protein stability determinants. Academic curiosity is now tackling the issue of the relationship among stability/activity/flexibility of thermophilic proteins. The main question is whether in enzymes stability and activity are strictly related or not. The answer is not clear-cut. An interesting observation from our experiments is that the enzymatic activity of theΔN21 mutant is higher than that of the WT enzyme at low temperatures. Previously, we have determined the crystal structure of the mutantΔN21 mutant (2QZP).We found that the B factors of the mutant are significantly higher than those of the wild type enzyme in most parts of the structure; the averaged B factors rise from19.4 ?2 for the wild type to 28.4 ?2 for the mutantΔN21. The analysis of the structural features of both wild type and the truncated mutant reveals that there is a decrease in hydrogen bonds and salt bridges in both domains of the mutant, although there are no significant changes in the static structures between the wild type and theΔN21 mutant. It is likely that the increased flexibility of the active site, which is located at the interface of the two domains, is caused by the disruption of ionic interactions and hydrogen bonds. The conformation flexibility of the wild type and theΔN21 mutant was also investigated by the FIRST method. Compared with the wild type, the mutant ApAPH-ΔN21 exhibited flexible changes, including propeller I, II, III and residues 497–502 and 553–567. Especially, N-terminal truncation destroyed the original secondary structure of residues 559–567, replaced with high flexible loops which are close to the active site. Such flexibility changes can contribute to the high activity at the low temperature range, but is also linked to a reduced stability of the molecular edifice.更多还原