【作者】 东升；

【导师】 张玉忠； 陈秀兰；

【作者基本信息】 山东大学 ， 微生物学， 2014， 博士

【摘要】 褐藻酸主要是由褐藻或者特定种属的细菌产生的一类多糖。它们都是由p-D-甘露糖醛酸(β-D-mannuronate, M)与其差向异构体α-L-古罗糖醛酸(a-L-guluronate, G)两种残基通过1,4糖苷键相连而成的非支链共聚物,两种残基可以均聚,形成.M片段(polyM)或者G片段(polyG),或者交替排列形成MG片段(polyMG)。褐藻酸的降解都是依靠裂解酶类通过β-消除反应进行,而没有水解酶类参与。褐藻酸裂解酶能够降解某些致肺纤维囊肿疾病(cystic fibrosis, CF)细菌形成的褐藻酸多糖膜,因此可作为辅助治疗该类疾病的有效措施。最近,褐藻酸裂解酶也被用于从海藻制备生物乙醇的研究过程中。因此,褐藻酸裂解酶有重要的应用价值。在CAZY数据库(Carbohydrate-Active enZYmes Database)中,褐藻酸裂解酶分布于裂解酶(Polysaccharide Lyase, PL)5\6、7、14、15、17、18家族中。褐藻酸裂解酶通过p-消除反应打开糖苷键,在C4和C5之间形成不饱和的双键。褐藻酸裂解酶已经从多种类型的物种中分离到,例如海藻、海洋软体动物、细菌、真菌甚至病毒等。北极地区是地球上的极端环境之一,生活在北极的生物形成了适应极端环境的独特机制,因此北极地区是开发利用新型生物资源的宝库。虽然目前已经有很多从附着在海带上的微生物中分离筛选褐藻酸裂解酶的研究报道,但是还没有对北极褐藻酸裂解酶产生菌株的研究。本论文以北极海带样品为研究材料,进行了产生褐藻酸裂解酶菌株的筛选和多样性分析,为进一步筛选新型适冷褐藻酸裂解酶奠定了基础。随着研究的深入,越来越多的褐藻酸裂解酶被解析了蛋白质结构,它们的结构与功能的关系也不断被阐明。PL18家族是裂解酶家族中比较新的一个家族,对该家族的研究还较少,还基本没有关于该家族褐藻酸裂解酶的底物识别机制、催化机制以及成熟机制的报道。Pseudoalteromonas sp. SM0524是我们从烟台近海分离到的一株高产褐藻酸裂解酶的菌株,该菌所产的PL18家族褐藻酸裂解酶aly SJ02是一种高效的双功能酶。该菌还分泌另一种PL7家族褐藻酸裂解酶aly SJO1。本论文对PL7家族酶aly SJO1的酶学性质和底物降解特异性机制、PL18家族酶aly SJ02的成熟机制、底物识别和催化机制进行了研究。(1)北极海带样品产褐藻酸裂解酶菌株的筛选和多样性分析从取自北极地区的6个海带样品上,通过富集培养和涂布筛选平板,共挑取了74株菌进行划线纯培养,然后在16S rRNA基因序列分析基础上进行分子系统发育分析。结果表明它们是65株不同的菌,Psychrobacter、Psychromonas和Polaribacter是其中的优势属,占筛选到可培养菌株的75%以上。进一步从中筛选到了21株可以分泌褐藻酸裂解酶的菌株。系统发育分析表明这21株产酶菌株分布在5个属中,分别为Psychrobacter、Winogradskyella、 Psedoalteromonas、Psychromonas和Polaribacter。我们首次发现来源于Psychrobacter、Winogradskyella、Psychromonas和Polaribacter属的细菌能分泌褐藻酸裂解酶。这些菌最适生长以及分泌褐藻酸裂解酶的温度大部分低于25℃,在低温环境下可以生长良好,表现出了明显的适冷特性。其中某些菌株的最大产酶能力超过了200U/mL。因此,北极的海带样品是筛选产适冷的褐藻酸裂解酶菌株的良好原材料。这些研究结果为进一步筛选新型适冷褐藻酸裂解酶奠定了基础。(2)PL7家族褐藻酸裂解酶aly SJO1的酶学性质和底物特异性机制研究aly SJO1是Psedoalteromonas sp. SM0524编码的一个褐藻酸裂解酶基因。序列分析表明,该酶属于PL7家族,与已经研究过的来自Vibrio sp. QY101的AlyVGI相似性最高,仅有41%的序列相似性,表明该酶的氨基酸序列具有新颖性。利用Escherichia coli异源表达系统,克隆表达了aly SJO1。对aly SJO1的性质分析表明,aly SJO1的最适酶活温度为30℃;并且该酶对热敏感,超过30℃的温度条件活性丧失很快,45℃条件下15min基本完全失活；该酶的Tm(Melting temperature)值仅为37℃,对SDS等变性剂的耐受能力很差,这表明aly SJ01的结构柔性较强,稳定性较差。因此,以上结果表明aly SJO1是一种适冷酶。aly SJO1的最适pH为8.5;可以被NaCl激活,在0.6M(3.5%)的NaCl条件下酶活可以提高6倍,并具有很好的耐盐能力。这些性质表明,作为一个海洋来源的酶,aly SJO1已经很好地适应了海水碱性、高盐、低温的环境。对酶解产物的分析表明,aly SJO1酶解产物主要为二聚体和三聚体,也有少量的四聚体以及更高聚合度的产物。另外酶解产物中也有少量的单体存在,aly SJO1是否具有外切酶活性尚需进一步验证。对aly SJO1的底物特异性分析结果表明,该酶对PM片段的酶活非常高,但是对PG片段基本没有降解活性,表现出了非常好的PM降解特异性。在已经研究报道的褐藻酸裂解酶中,AlyVGI与aly SJO1的序列相似性最高,但是底物特异性却不同。我们对这两者进行序列比对,发现这两个酶都有PL7家族典型的保守区,但是覆盖在催化腔的lid-loop上的差异非常大。另外,在催化腔附近也有一些氨基酸序列的差异,我们据此设计了突变。分别测定了aly SJO1突变体对PM和PG片段降解活性的改变。结果表明,PL7家族褐藻酸裂解酶aly SJO1底物降解特异性与催化腔中的保守的关键氨基酸关系不大,与降解偏好性有关的氨基酸多分布在催化腔周围以及lid-loop上,而这些氨基酸在该家族序列上不是很保守。因此,催化腔周围以及lid-loop上这些不保守氨基酸赋予了该家族酶多样的底物偏好性。(3) alySJ02的成熟机制及其N端结构域功能的研究序列分析表明,aly SJ02的全基因包含四个部分：信号肽(Met1-Ala31)、N端结构域(ND)(Ala32-Gly155)、linker (Ser156-Ser173)和催化结构域(CD)(Thr174-Asn400)。PL18家族的ND在酶的成熟过程中被切除,成熟酶仅含有CD。我们在Escherichia coli中单独表达aly SJ02的ND和linker共含有142个氨基酸的蛋白,通过实验验证了该结构域蛋白具有多糖吸附功能。但是在酶原中,该结构域的存在并没有增加酶对底物的亲和力,不能发挥底物吸附功能。另外,在Escherichia coli中单独表达的催化结构域虽然也有催化活力,但是与成熟酶相比,其催化活性要低20%左右。利用两个载体在Escherichia coli中共表达ND和CD,结果表明这两个结构域蛋白能够通过相互作用结合到一起,形成一个复合物。但是,如果在不同的Escherichia coli细胞中分别表达ND和CD,将折叠好的两者蛋白混合,他们不再有相互作用,不能形成一个复合物。这在天然条件是非常重要的,可以保证成熟过程中酶的linker位置被切断以后两者能顺利分离,使aly SJ02的CD能形成成熟酶的形式。因此,生化证据证明了ND可以促进CD的折叠成熟。为了进一步分析ND促进CD折叠成熟的分子机制,我们对异源表达的CD蛋白和带有ND的前体进行了结晶。PDB数据库中来于Psedoalteromonas sp.272的褐藻酸裂解酶(PDB.1J1T)与aly SJ02成熟酶序列只有一个氨基酸残基差异,我们以其为模型,分别同源模建了aly SJ02成熟酶的结构、解析了单独表达的CD和带有ND的前体中的CD的结构。通过结构比较分析,我们发现带有ND的前体中的CD的结构中关键氨基酸的构象与成熟酶是完全一样的,而在没有ND条件下,CD的折叠是不规范的。不对称单位的两个分子中,一个分子的构象与成熟酶基本一致,而另一个分子的很多保守氨基酸的空间构象与成熟酶差别非常大。因此,生化证据和结构分析都表明,在aly SJ02的成熟过程中,ND可以促进催化结构域的正确折叠,特别是保证催化活性中心关键氨基酸的正确构象。这些实验结果表明,N端结构域在PL18家族褐藻酸裂解酶的成熟过程中可能起着分子内伴侣的功能。(4)PL18家族褐藻酸裂解酶aly S J02的底物识别和催化机制研究aly SJ02蛋白的基本结构是由两层反向平行的p折叠片层卷曲围绕而成,与已经提交PDB数据库的PL7和PL18家族的褐藻酸裂解酶的结构非常类似。结构和原子吸收光谱分析表明,aly SJ02蛋白中含有一个钙离子,钙离子虽然远离催化活性中心,但用EDTA螯合出酶中的钙离子会导致aly SJ02的催化活性降低50%左右,因此钙离子虽然不参与到催化反应过程中,但是可以稳定酶结构,保持酶的高催化活性。在褐藻酸裂解酶催化腔上方普遍存在一种由2个loop组成的类似盖子的结构,这些loop称为lid-loop。我们对aly SJ02的lid-loop进行分子动力学模拟,结果表明,aly SJ02的2个lid-loop之间的距离可以从大约3A增大到11A左右,表现出了明显的开与合两种状态。通过双突变N216C/T263C在2个lid-loop间形成二硫键,会导致其柔性丧失,结果也使酶的活性完全丧失；加入还原剂DTT破坏二硫键使lid-loop的柔性逐步恢复,酶的催化活性也会逐步部分恢复。因此,lid-loop对aly SJ02识别结合底物非常重要。为了分析aly SJ02对底物的识别和催化机制,我们建立了aly SJ02与一个4聚甘露糖醛酸寡糖的复合物的Docking模型。通过对aly SJ02与底物的Docking模型的分析,确定了酶与底物的结合位点,其中,糖醛酸残基A-1被氨基酸残基Gln355和Lys364识别。在位点S+1,Gln257和His259识别结合糖残基A+1的羧基,Thr263识别结合02, Asn216识别结合03, Thr353识别结合04。糖残基A+2的羧基被Arg219和Lys349识别结合,03和04被His259识别结合。在位点S+3,羧基被Lys223和Thr347识别。对这些位点进行了突变验证,S+1和S+2位负责识别羧基的氨基酸非常保守,突变会导致酶完全失活,与糖残基的羟基作用的氨基酸突变后,会保留部分催化活性；S-1和S+3位参与了底物的识别结合过程的氨基酸用Ala置换突变后,酶的活性丧失,但是置换为性质接近的氨基酸时,可以保留酶的部分催化活力。有活性突变体的Km值增大,表明突变使酶与底物的亲和力降低。因此,aly SJ02的底物识别结合位点主要是在S+1和S+2,这些位点的氨基酸主要是识别结合多糖的羧基,而与糖环上的羟基氧原子作用的氨基酸的保守性较差。S-1和S+3位点的氨基酸对底物识别也非常重要,但是比S+1和S+2位点的重要性稍弱。aly SJ02对底物的识别机制决定了对底物进行充分酶解后的产物应该是以二聚体和三聚体为主,与之前的报道的实验结果一致。根据结构和突变分析结果,我们分析了aly SJ02催化褐藻酸裂解的反应机制。保守氨基酸Arg219, Lys223, Gln257, His259和Lys349可以识别结合底物,特别是结合稳定底物的羧基基团：在反应过程中,这些氨基酸形成的正电势的催化中心对碳负离子的稳定也非常有利。氨基酸Tyr353与A+1的C5的距离为3.1A,与04的距离是3.3A,该氨基酸突变会导致酶完全失活,其他氨基酸离C5和04的距离都较远(>4A),因此推测Tyr353既可以作为催化碱又作为催化酸。因此,根据实验结果,我们提出了aly SJ02的催化过程和机制：首先,在lid-loop开放状态下,酶与底物结合；Tyr353从A+1的C5位上获取质子,该位置形成碳负离子,保守氨基酸形成的正电势催化腔对其稳定十分有利；然后Tyr353又作为质子供体,将质子转移给04,导致糖苷键断裂,同时在C4与C5之间形成不饱和的双键。这是首次解析PL18家族褐藻酸裂解酶的成熟机制和底物识别与催化机制,研究结果为进一步研究该家族褐藻酸裂解酶的结构与功能及其应用开发奠定了基础。更多还原

【Abstract】 Alginate is synthesized as a cell wall component by brown algae and also as an exopolysaccharide by certain bacteria. This polysacchatide is a natural liner polysaccharide composed of (1,4)-linked β-D-mannuronate(M) and its C5epimer, a-L-guluronate (G). These uronic acids are arranged in block structures which may be homopolymeric M block (polyM), and homopolymeric G block (polyG), and heteropolymeric blocks with random arragement of both monomers (polyMG) Alginate lyase catalyze the degration of polysaccharides by β-elimination reaction. Alginate lyases hold promise as biochemicals for removing bacterial biofilm alginate which functions as an important virulence factor during lung infections in cystic fibrosis patients. More recently, alginate lyases have been expected to become potential enzymes in the bio-energy generation from alginate. Thus, alginate lyases are important enzymes in a broad spectrum of biological roles and applications. Alginate lyases are classified into7families (family PL5,6,7,14,15,17,18) in the Caborhydrate-Active enZYmes (CAZy) data base. These lyases catalyze the degradation of alginate by a β-elimination of the4-O-glycosyl bond accompanied by the formation of a double bond between C-4and C-5and the production of4-deoxy-L-erythro-hex-4-ene pyranosyluronate at the nonreduceing end of the resulting oligosaccharides. Alginate lyases have been isolated from various sources, namly marine algae, marine mollusks, fungi, bacteria, bacteriophages, and viruses.Various alginate lyases are discoveried from marine bacteria that associated with brown alga. The Arctic Ocean is the most extreme ocean regarding the seasonality of light and its year-round existing ice cover. A large number of unique life forms are highly adapted to the extreme environment of the Arctic Ocean in their ecology and physiology. Brown alga growing in the intertidal zone of the Arctic Ocean is a potential resource for the discovery of new bacteria producing novel alginate lyases, which, however, have never been investigated. In this dissertation, the cultivable alginate lyase-excreting bacteria associated with the Arctic brown alga Laminaria were isolated and the diversity of them were further studied, which constituted a groundwork for mining novle cold-adaped alginate lyases from these Arctic bacterial resources.Up to now, the crystal structures of some alginate lyases have been detrermined. The relationships between structure and function of these enzymes have been well demonstrated. PL18is a relatively new family and mechanisms on the catalysis, substrate recognization and mature of enzymes in this family were therefore not clear. A marine bacterium with high alginate-lyase production, named Psedoalteromonas sp. SM0524, was preiously isolated from Yantai, China in our Lab. Two alginate Iyases are secreted by this bacterium, namely aly SJO1and aly SJ02. aly SJ01is belonged to PL7while aly SJ02, a bifunction alginate lyase with high activities,, is a member of PL18. In this dissertation, the enzymatic properties and mechanisms of substrate specificity for the PL7alginate lyase aly SJO1were firstly studied; crystal structures of family PL18aly SJ02were then determined, through which mechnism of substrate recognition and the function of N-terminal domain are also studied..The results are as following.(1) Isolation and diversity analysis of alginate lyase-extracting bacteria from Arctic Laminaria.A total of74isolates were isolated from6Arctic Laminaria samples and they were further purified and subjected to16S rRNA gene amplification. According to an alignment of the16S rRNA gene sequences, a total of65different strains were isolated from the Arctic Laminaria samples. Phylogenetic analysis based on the16S rRNA gene sequences showed that the isolated Lam/nana-associated bacteria belonged to nine genera. Psychrobacter (33/65) were the most predominant group. Psychromonas (10/65) and Polaribacter (8/65) also showed some preponderance.21algiante lyase-excreting bacteria were further screened from the65 Lammarria-associated bacteria and phylogenetic analysis based on the16S rRNA gene sequences revealed that they were belonged to5genera, namely Pseudoalteromonas, Psychrobacter, Winogradskyella, Psychromonas and Polaribacter. Alginate lyase-excreting marine bacteria so far reported include Alginovibrio, Alteromonas, Beneckea, Halomonas, Photobacterium, Pseudoalteromonas and Vibro. Besides Pseudoalteromonas, it was first found that members of genera Psychrobacter, Winogradskyella, Psychromonas and Polaribacter excreted alginate lyases. Among these Arctic algiante lyase-excreting strains, the optimal temperatures for growth and algiante lyase production of many strains were lower than25℃, showing that they were psychrophilic bacteria. The alginate lyases excreted by11strains showed the highest activity at20-30℃, indicating that these alginate lyases were cold-adapted enzymes. Moreover, some strains showed high extracellular alginate lyase activity levels around200U/mL. These results suggest that the algiante lyase-excreting bacteria from the Arctic alga Laminaria may be good materials for studying bacterial cold-adapted alginate lyases.(2) Enzymatic characterization and substrate specificity mechanism study of the PL7alginate lyase aly SJ01A novel alginate lyase named aly SJ01was cloned from the marine strain Pseudoalteromonas sp. SM0524. Sequence analysis showed that aly SJ01belonged to the PL7family and had only about41%highest identity to a PL7characterized alginate lyase, AlyVGI from Vibrio sp. QY101. aly SJ01was further expressed in Escherichia coli and the enzymatic properties of this enzyme were studied. The optimal temperature of aly Sj01was at around30℃. aly SJ01was rapidly inactivated above30℃and about80%of enzyme activity was lost on preincubation at40℃for15min, indicating the low thermal stability of this lyase.. The apparent melting temperature (Tm) of Aly SJ01determined by Circular Dichroism (CD) was37℃. These results indicated that aly SJ01was a cold-adaptive alginate lyase. The optimal pH of aly SJ01is at8.5; its activity was increased6times by adding0.6M NaCl, a value approximately equal to that in seawater. All the above results suggested that aly SJO1is active and thereby play an important biological role in cold, slightly alkaline and salty marine environments. PolyM, polyG and alginate were respectively reacted wih aly SJO1and the resultant oligosaccharides with different degrees of polymerization were analyzed by TLC. Results from the TLC analysis indicated that enzymatic products were mainly dimmer, trimmer and tetramer with small amounts of monermers and some larger oligosaccharides. The substrate specificity result indicates that aly SJ01had a preference for poly M substrates rather than poly G and poly MG. Despite of the the highest identity with alginate lyase AlyVGI, aly SJ01exhibited different substrate specification from AlyVGI. To better understand the substrate specificity of aly SJ01, amino acids sequence alignments were performed and site-directed mutagenesis were conducted. Analysis focused on mutations in aly SJ01with significant effect on specificity or activity. In summary, conserved amino acids in the active center show little relations to substrate specificity while some introduced mutations in Loop1and Loop2and around the active center apparently increased enzyme activity for polyG with decreased activity towards polyM. Hence, the substrate specificity of aly SJ01seemly results from the unconserved residues around the active center.(3) N-terminal function analysis of PL18alginate lyase aly SJ02The precursor of aly-SJ02contains a signal peptide (Metl-Ala31), an N-terminal domain (Ala32-Gly155)(ND), a linker (Ser156-Ser173) and a PL18catalytic domain (CD)(Thr174-Asn400). Mature aly-SJ02only contains the CD while ND was cut off. The N-terminal domain of aly-SJ02showed alginate binding ability when it was recombinantly expressed individually. However, it showed no binding ability in the precursor of aly SJ02. The enzymatic activity analysis showed that the activity of CD toward alginate was approximately20%lower than that of mature aly SJ02. When the The N-terminal and catalytic domains domain of aly-SJ02were co-expressed with two different vectors in an E. coli cell, the ND and the CD could form a protein complex by interaction, while the folded ND and CD expressed in different E. coli cells could not. To better eluciate that how the ND affects the CD folding, the CD crystal structures from different folding process, namely expressed individually(r-CD) and expressed in precursor(P-CD) were solved. Taking the Psedoalteromonas sp.272alginate lyase (PDB:1J1T) as model, the crystal structure of aly SJ02(M-CD) was obtained by homology modeling. A detail comparison of conformation of these conserved residues in the active centers of r-CD, P-CD and M-CD was preformed. These conserved residues in P-CD and M-CD have the same conformation, suggesting that P-CD has folded correctly. For r-CD, while those residues in molecule B in the crystal unit shares almost the same conformation as those in P-CD (except a tiny swing between Arg219and Tyr353in the two molecules), molecule A differs greatly from P-CD in their conformation. This indicates that, without the presence of the ND, the CD of aly-SJ02may fold disorderly, thereby affecting its activity toward alginate. Base on the above results, it was proposed that the N-terminal domain has evoluted a new function faciliating the folding of catalytic domain.(4) Substrate recognition and catalysis mechanism studies of PL18alginate lyase aly SJ02The PL18alginate lyase aly SJ02had a P-sandwich fold consisting of two β-sheets creating a deep active cleft which was covered by two flex loops. This crystal scaffold was also common shared by the family PL7and PL18alginate lyases. Atomic absorption spectroscopy confirmed that a Ca2+is present in aly-SJ02. The chelate site of Ca2+is far away from the reaction center, suggesting that this metal ion is not directly involved in the polysaccharide degrading reaction. Deprivation of Ca2+from aly-SJ02by EDTA could result in a50%reduce in enzymatic activity. This indicates that the Ca2+in aly-SJ02is important for keeping the enzymatic activity though it does not participate directly in the catalytic reaction. It has been found that alginate lyases usually have loops covering the active cleft. To investigate whether the lid loops of aly-SJ02have a gating function in substrate entry and product release, we performed an Molecular Dynamics simulation for aly-SJ02P-CD structure, with a particular focus on these two loops. Distance measurement of the side chains of Asn214and of Thr263suggests that the space between the loops can increase to11.5A in the "open" state from3.2A in the "closed" state, which makes it possible for an alginate molecule entering the substrate-binding pocket of aly-SJ02. A mutant Asn214Cys/Thr263Cys with Asn214and Thr263being replaced by cysteine residues was constructed to introduce a rigid interaction between loop1and loop2by forming a disulfide bond. This mutation almost completely abolished the activity of aly-SJ02, and a reduction of this disulfide bond by DTT would increase the mutant activity significantly. These result indicates that keeping the flexibility of the lid loops is essential for substrate entry into the substrate-binding pocket of aly-SJ02.Uronic acid residue A-1is accommodated at subsite-1by residues Gln355and Lys364. At subsite+1, the carboxyl group of the saccharide residue A+l is recognized by Gln257and His259, O2by Thr263,03by Asn216, and O4by Thr353. At subsite+2, the carboxyl group is recognized by Arg219and Lys349, and03and O4by His259. At subsite+3, the carboxyl group of A+3is recognized by Lys223and Tyr347. Amino acid substitutions of conserved residues at subsites+1and+2resulted in inactivation of the enzyme while some mutation of those located at subsites-1and+3still reserved enzyme activities. The Km values of the active mutants were all increased, indicating these mutations decreased the affinity of aly-SJ02to the substrate. The residues located at subsites+1and+2are more rigidly conserved than the others, indicating the substrate recognition and binding mainly happened at subsite+1and+2, while residues at the subsites-1and+3have some relatively weak interactions which is consistent with the previous results that aly-SJ02depolymerized alginate and released dimer and trimer mainly.Based on Docking model of the aly-SJ02-oligosaccharides complex and our mutation assays, the catalytic mechanism of aly-SJ02for alginate degradation is proposed. The distance between C5and Tyr353is3.1A, and that between04and Tyr353is3.3A. Mutations of Tyr353resulted in inactivation of the enzyme. Tyr353is function as a catalytic base and acid, which can obtain a proton as a catalytic base from the C5of A+1and give proton to04as a catalytic acid. And residues Arg219, Gln257, His259and Lys349that around subsite+1and+2, stabilizing or neutralizing the negative charge of uronic acid, are quit crucial for substrate recognization and the catalytic reaction,To our knowledge, the above results are the first reports about the substrate recognition and catalytic mechanism of PL18alginate lyases, which will facilitate the studies on structure and function of alginate lyases in this family and provide a foundation for developing new applications of alginate lyases.更多还原