【作者】 李渊；

【导师】 孙宇辉； 瞿旭东；

【作者基本信息】 武汉大学 ， 分子生物学与生物化学， 2018， 博士

【摘要】 天然产物是人类药物的重要来源。随着研究的深入,新发现的具有新颖结构和活性的天然产物越来越少,而且抗生素的滥用导致细菌的耐药性问题越来越严重。因此,阐明天然产物特别是抗生素的生物合成途径,并对其结构进行改造,增强其药效或成药性,是未来新型高效的抗生素研究的重要方向。本研究是以氨基糖苷类和聚酮类化合物为研究对象,利用结构生物学的方法对其生物合成途径中的关键酶进行了详细的探究。第一部分内容为庆大霉素合成途径中转氨酶GenB1及异构酶GenB2的结构生物学研究。庆大霉素是一种多组分混合物,每个组分的毒性和抗菌活性不同,获得其单组分是提高其用药功效的必要手段,GenB1和GenB2负责绛红糖胺C’-6位氨基化,特别是GenB2的异构化作用,是决定生成C2和C2a的关键酶。阐明它们的结构和机理,对获得庆大霉素单一组分非常重要。首先表达出高纯度的GenB1和GenB2蛋白,经过蛋白晶体的初筛和优化,获得了高分辨率的晶体,通过分子置换解析了它们的母体结构,同时,我们通过共晶的方法获得了 GenB2与底物类似物G418的复合物结构,GenB1和GenB2是典型的Type I类PLP依赖的酶,以二聚体的形式发挥作用。PLP位于C端功能域中间和PLP结合功能域组成的活性口袋内,在其中有三个保守的残基Asp、Thr和另一个单体分子中的Thr结合PLP,把PLP稳定在活性口袋。与其它Type I类酶相比,GenB1和GenB2存在一个大的活性口袋容纳PLP和三个糖环组成的底物,且在活性口袋中存在大量的亲水残基与小分子相互作用。通过比对GenB1和GenB2的活性口袋发现:它们的活性口袋非常保守,仅有的差异在于GenB1的N端功能域上有一个极性残基Arg71,而在GenB2中为Phe62,此氨基酸的差异可能是两者功能差异的关键原因。对比其它的Type I类异构酶,发现GenB2活性口袋存在保守的Lys256/Tyr143残基,推测GenB2的异构化机理也采用“two-base mechanism”的方式。第二部分内容为具有底物宽泛性的酰基转移功能域SpnD-AT的结构机理研究。聚酮合酶PKS模块中的酰基转移功能域AT是识别不同延伸单元的“大门”,通过改造AT可以获得具有不同活性的非天然化合物。SpnD-AT是一种具有底物宽泛性识别能力的酰基转移功能域,能特殊的招募芳香型延伸单元,突破了 AT只能识别简单烷基延伸单元的限制。首先表达出纯化出高纯度的蛋白SpnD-AT,经过蛋白晶体的初筛和优化,成功获得分辨率为为2.35 A的数据,通过分子置换解析了 SpnD-AT的母体结构。然后用两种底物(benzylmalonyl-SNAC 和 pentynylmalonyl-SNAC)浸泡母体晶体,获得了两个复合物结构。SpnD-AT的结构具有典型的酰基转移酶特征,由一个大的α/β水解结构域与一个小的铁氧还原蛋白结构域组成,底物位于由α/β水解功能域和铁氧还原蛋白功能域的loop264-272构成的口袋中,周围有三个保守的催化作用残基:Ser173、Arg198、His271。底物为S构型,其疏水侧链被一些疏水残基包围,其中Phe145的侧链苯环与底物的苯环和炔基形成π-π相互作用,是识别芳香型底物的重要残基。将SpnD-AT与红霉素Ery-AT5进行结构比对,显示SpnD-AT有一个大而疏水的活性口袋,而在Ery-AT5中三个保守的残基Tyr278、Ser280和Gln150影响了活性口袋的大小和疏水性,说明这三个保守残基对底物的选择性至关重要。把红霉素Ery-AT6的三个保守的残基Tyr278、Ser280和Gln150突变为小而灵活的残基,并对不同大小的底物进行筛选,发现Y278G-S280G对C5、C7和benzyl底物的招募能力大大增加,同时Y278A-S280A对C3底物也具有比较强的催化活性。此结果扩大了 Ery-AT6对较大体积侧链底物的选择,为红霉素及其它聚酮化合物产生菌株的理性改造提供了了基础和方向。更多还原

【Abstract】 Natural products are an important source of human medicine.however,discovering new natural products with novel structures and activities are becoming less frequent,and the abuse uses of antibiotics has led to a growing problem of bacterial resistance.Therefore,clarifying the biosynthetic pathway of natural products,especially antibiotics,is imperative to improve their efficacy or drug resistance.The research object of this paper is aminoglycosides and polyketides.We have made an intensive study of the key enzymes in the biosynthetic pathways with the structural biology.The first part of this paper is to study the structural biology of transaminase GenB 1 and isomerase GenB2 in Gentamicin biosynthesis pathway.Gentamicin is a mixture with various component.The toxicity and antibacterial activity of each component are different.Obtaining the monocomponent is essential to improve the efficacy.GenB1 and GenB2 are responsible for the amination at the C’-6 position,especially the GenB2,is the key enzymes to generate C2 and C2a.Determination of their structure and mechanism is quite significant for obtaining the monocomponent of gentamicin.Firstly,we acquired high-purity GenB1 and GenB2 proteins.high-resolution crystals were obtained by crystal screening and optimization,their structures were solved by molecular replacement,and we obtained the structure of GenB2 complex with the substrate analog G418.GenB1 and GenB2 are typical Type I PLP-dependent enzymes that act as a dimer.The PLP is located in the active pocket between a small C-terminal domain and a large PLP-binding domain,where existed three conserved catalytic residues(Asp,Thr,and a Thr from in another monomer molecule)stabilizing the PLP.Compared with other Type I enzymes,GenB1 and GenB2 have a large active pocket that contains PLP and three sugar ring substrates,and there are numerous hydrophilic residues in the active pocket that interact with substratethe residues of the active pockets of GenB1 and GenB2 were found to be very conserved.The only difference is that GenB1 has a polar residue Arg71 on the N-terminal domain,while orresponding residues are changed as Phe62 in GenB2.The discrepancy may be the key reason for their functional distinct.Comparing to other Type I isomerase revealed a conservative Lys256/Tyr143 residue in the GenB2 active pocket,meaning that the GenB2 also adopts the "two-base mechanism".The second part of the dissertation is about the structural basis of the broad-selective acyltransferase domain SpnD-AT.The acyltransferase domain AT in the polyketide synthase(PKS)module is the "gateway" to identify different extender units,and modifying AT can obtain numerous non-natural compounds with different activities.SpnD-AT is an acyltransferase domain with broad-selective for various substrates,and can specifically recruit aromatic extender unit,breaking the limitation that the traditional ATs can only select limited malonyl thioestersWe first expressed the SpnD-AT and obtained the high-purity protein.The regular shape crystals were obtained by crystal screening and optimization,and collected a set of reflection data at 1.6 A.The structure of SpnD-AT was solve.d by molecular replacement.Then we soaked the crystals with two substrates(benzylmalonyl-SNAC and pentynylmalonyl-SNAC)and obtained structures complex with two substrates.The structure of SpnD-AT is Similar to other canonic AT domains,consisting of a large α/βhydrolase fold and a small ferredoxin.The substrate located at a hydrophobic interface of the α/β-hydrolase and ferredoxin subdomains where existed three conserved catalytic residues(Ser173,Arg198,and His271).The extender unit formed a covalent link with OH of the Catalytic residues Ser,and the a-carbon present an S configuration.The side chain of Phe145 formed strongπ-π interaction with the phenyl and the alkynyl group of the substrate and regarded as an important residue for the recognition of aromatic extender units.We compared the structure of SpnD-AT with erythromycin Ery-AT5,and found that SpnD-AT has a large hydrophobic active pocket which is very important for building AT specificity and activity.in Ery-AT5,there are three conserved residues Tyr278,Ser280,and Gln150 that affect the size and hydrophobicity of active pocket,which demonstrates the three residues were identified critical for restricting their substrate specificity.Engineering the residues Tyr278 and Ser280 of Ery-AT6 to flexible residues can either reverse or broaden the substrate specificity.This study pave a solid way to introduce diverse structural varieties into the polyketide carbon scaffold.更多还原