【作者】 周韵；

【导师】 孙莲莉；

【作者基本信息】 浙江大学 ， 药学（专业学位）， 2021， 硕士

【摘要】 选择性不对称还原α,β-不饱和羰基化合物的羰基或烯键,可获得手性烯醇或饱和羰基化合物,在药物合成和天然产物结构修饰领域具有重要的应用价值。但由于羰基和烯键的键能较为接近,利用化学方法较难控制α,β-不饱和羰基还原反应的区域选择性和立体选择性,目前主要利用过渡金属及其有机配合物作为催化剂,但过渡金属价格昂贵、易污染,且其配合物合成步骤复杂,工业化应用困难。与化学法相比,酶催化羰基/烯键还原无区域选择问题,且普遍具有立体选择性强、反应条件温和、产物单一易纯化、高效环保等优势,更具有工业化开发的前景。Perakine还原酶(Perakine Reductase,PR)来源于药用植物蛇根木(Rauvolfia serpentina),为醛酮还原酶(AKR)超家族成员,以NAD(P)H为辅因子,催化还原多种结构类型的羰基类化合物,底物谱广、立体选择性强、稳定性好、异源表达量高、易纯化制备,具备良好的工业化基础,但目前其催化效率无法达到工业应用水平,仍需筛选羰基还原高效突变体。根据AKR超家族中羰基还原酶和烯键还原酶突变催化中心特定位点可实现两者活性的转化的特性,有望将PR定向突变为烯键还原活性,提升PR的应用价值,并为烯键还原提供性能良好的生物催化剂。本论文在Perakine还原酶复合物晶体三维结构的基础上,利用氨基酸序列比对和计算机分子模拟,确定126位组氨酸和127位精氨酸为PR的底物羰基结合关键位点,并采用饱和突变的方法对上述两个位点进行突变,以亚苄基丙酮为底物,利用高效液相色谱检测产物收率和底物转化率,确定突变体羰基或烯键还原相对活性。经测序验证,成功获得126位和127位突变体各19种。在PR的126位突变体中,除了4种突变体失活外,其他15种126位突变体全部转变为烯键还原活性,烯键活性最强的突变体为H126G;127位突变体的活性情况则较为复杂,9种突变体仍保留羰基还原活性,6种突变体转换为烯键还原活性,3种突变体则同时具有羰基和烯键还原活性,1种突变体无还原活性,但与野生型相比,所有127位突变体的羰基还原活性均显著降低,其中羰基还原活性最高的R127L突变体的羰基还原相对活性也仅为野生型的5.7%,R127M是烯键还原活性最强的突变体,相对活性比H126G突变体高～40%;将126位和127位突变体中烯键活性较强的突变进行组合双突变,但双突变体的烯键还原活性显著降低。PR突变体催化动力学研究表明突变体和野生型与底物亲和力无明显差异。为进一步阐明126和127位氨基酸改变PR催化功能的机理,我们选择了126位和127位突变体中烯键还原活性较好的突变体进行结构生物学研究,摸索PR突变体-辅因子-底物复合物结晶条件,但目前为止未能获得复合物晶体。在结晶未果的情况下,我们采取计算机分子对接和动力学模拟方法,对PR突变体的催化机理进行研究。分别选取具有代表性的羰基还原活性、烯键还原活性、羰基烯键双还原活性的突变体与底物分子亚苄基丙酮进行对接及动力学模拟实验,结果表明突变体中底物分子的羰基和烯键与辅因子烟酰环之间相对位置决定了还原选择性。本论文的研究成功实现了PR羰基/烯键还原选择的定向突变为选择性还原α,β-不饱和羰基化合物提供催化性能优良的新型生物催化剂,显著提升了PR的应用价值。更多还原

【Abstract】 Selective asymmetric reduction of the carbonyl or alkene bonds ofα,β-unsaturated carbonyl compounds can lead to chiral enols or saturated carbonyl compounds,which have important applications in the fields of drug synthesis and structural modification of natural products.However,since the bond energies of carbonyl and alkene bonds are relatively close,it is difficult to control the regioselectivity and stereoselectivity of α,β-unsaturated carbonyl reduction reactions using chemical methods.Currently,transition metals and their organic complexes are mainly used as catalysts,but transition metals are expensive and easily contaminated,and their complex synthesis steps are complicated,making industrial applications difficult.Compared with chemical methods,enzyme-catalyzed carbonyl/alkene bond reduction has no regioselectivity problem,and generally has the advantages of strong stereoselectivity,mild reaction conditions,single and easy purification of products,high efficiency and environmental protection,etc.,which is more promising for industrial development.Perakine Reductase(PR)is derived from the medicinal plant Rauvolfia serpentina and is a member of the aldehyde and ketone reductase(AKR)superfamily,which uses NADPH as a cofactor to catalyse the reduction of various structural types of carbonyl Vcompounds.It has a broad substrate spectrum,high stereoselectivity,good stability,high heterologous expression,easy purification and preparation,and has a good basis for industrialization,but at present its catalytic efficiency cannot reach the level of industrial application,and still needs to be screened for efficient carbonyl reduction mutants.Based on the property that mutations in the catalytic centres of the AKR superfamily of carbonyl reductases and alkene bond reductases can convert the two activities,it is expected that targeted mutation of PR to alkene bond reduction activity will enhance the application of PR and provide a good performance biocatalyst for alkene bond reduction.In this thesis,based on the crystal 3D structure of the Perakine reductase complex,amino acid sequence comparison and computer molecular simulations were used to identify histidine at position 126 and arginine at position 127 as the key sites for substrate carbonyl binding of PR,and the above two sites were mutated by saturation mutagenesis,and benzylidene acetone was used as the substrate,and the product yield and substrate were detected by high performance liquid chromatography The relative activity of the mutant carbonyl or alkene bond reduction was determined by high performance liquid chromatography.The sequencing was validated and 19 mutants each at positions 126 and 127 were successfully obtained.Among the 126 mutants in PR,all but four were inactivated and all the other 15 mutants at position 126 were converted to alkene bond reduction activity,with the strongest alkene bond activity being H126G;the activity of the 127 mutants was more complicated,with nine mutants retaining carbonyl reduction activity,six mutants converted to alkene bond reduction activity,three mutants having both carbonyl and alkene bond reduction The carbonyl reduction activity of all 127 mutants was significantly lower compared to the wild type,with the highest carbonyl reduction activity,R127 L,having only 5.7 % of the relative carbonyl reduction activity of the wild type,and R127 M being the mutant with the strongest alkene bond reduction activity,with a relative activity ～40 % higher than that of the H126 G mutant.The catalytic kinetics of the PR mutant showed no significant difference in the affinity of the mutant and the wild type for the substrate.To further elucidate the mechanism by which amino acids at positions 126 and 127 alter the catalytic function of PR,we selected mutants with better alkene bond reduction activity at positions 126 and 127 for structural biology studies to investigate the conditions for crystallization of PR mutant-cofactor-substrate complexes,but so far we have been unable to obtain crystals of the complexes.In the absence of crystallization,we have adopted computerized molecular docking and kinetic simulation methods to investigate the catalytic mechanism of the PR mutant.The results show that the relative positions of the carbonyl and alkene bonds between the substrate molecule and the cofactor nicotinyl ring in the mutant determine the reduction selectivity.This thesis has successfully achieved a targeted mutation of the PR carbonyl/alkenyl bond reduction option to provide a novel biocatalyst with excellent catalytic performance for the selective reduction of α,β-unsaturated carbonyl compounds,significantly enhancing the application of PR.更多还原