非洲栖热腔菌(Thermosipho africanus)纤维寡糖磷酸化酶的结晶及衍射分析
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摘要
为探究非洲栖热腔菌(Thermosipho africanus)纤维寡糖磷酸化酶TaCDP底物宽泛性的结构基础,通过Ni离子亲和层析-阴离子交换层析-凝胶过滤层析3步法纯化TaCDP,采用气相扩散法筛选TaCDP晶体,利用X-射线晶体学方法对TaCDP的晶体结构进行了研究。结果表明,纯化后TaCDP蛋白的纯度高,聚集状态均一,在2. 2 mol·L~(-1)(NH4)2HPO4,0. 1 mol·L~(-1)CHES-NaOH,p H值9. 2,10. 0 g·L~(-1)polyvinylpyrrolidone K15条件下筛选到TaCDP与纤维三糖底物的复合物晶体。该晶体经同步辐射收集衍射数据后分辨率可达0. 28 nm,晶体空间群属于P4122,晶胞参数为a=b=9. 928 nm,c=56. 251 nm,表明1个不对称单位有2个TaCDP分子。
To elucidate the structural basis of the broad substrate specificity of the cellodextrin phosphorylase from Thermosipho africanus( TaCDP),the TaCDP was purified with metal chelate afnity chromatography-anion exchange chromatography-size exclusion chromatography. Crystallization of TaCDP was performed by the vapour diffusion method and the crystal structures were analysed with the X-ray crystallographic method. The results showed that the purified recombinant TaCDP protein had high purity and uniform aggregation state. Composite crystals of TaCDP and fiber trisaccharide substrates were obtained under the conditions of 2. 2 mol·L~(-1)( NH4)2 HPO4,0. 1 mol·L~(-1) CHESNaOH pH 9. 2 and 10. 0 g·L~(-1) polyvinylpyrrolidone K15. The resolution of the crystal could reach0. 28 nm after collecting diffraction data by synchrotron radiation. The crystals belonged to space group P4122,with unit-cell parameters a = b = 9. 928 nm,c = 56. 251 nm,suggesting that there are two TaCDP molecules in one asymmetric unit.
To elucidate the structural basis of the broad substrate specificity of the cellodextrin phosphorylase from Thermosipho africanus( TaCDP),the TaCDP was purified with metal chelate afnity chromatography-anion exchange chromatography-size exclusion chromatography. Crystallization of TaCDP was performed by the vapour diffusion method and the crystal structures were analysed with the X-ray crystallographic method. The results showed that the purified recombinant TaCDP protein had high purity and uniform aggregation state. Composite crystals of TaCDP and fiber trisaccharide substrates were obtained under the conditions of 2. 2 mol·L~(-1)( NH4)2 HPO4,0. 1 mol·L~(-1) CHESNaOH pH 9. 2 and 10. 0 g·L~(-1) polyvinylpyrrolidone K15. The resolution of the crystal could reach0. 28 nm after collecting diffraction data by synchrotron radiation. The crystals belonged to space group P4122,with unit-cell parameters a = b = 9. 928 nm,c = 56. 251 nm,suggesting that there are two TaCDP molecules in one asymmetric unit.
引文
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[2] SHETH K,ALEXANDER J K. Purification and properties of beta-1,4-oligoglucan:orthophosphate glucosyltransferase from Clostridium thermocellum[J]. Journal of Biological Chemistry,1969,244(2):457-464.
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[13]王海波,叶雨佳,孟照辉.人甲状腺激素受体相互作用蛋白15的初步结晶实验及其在人组织表达的研究[J].中国生物工程杂志,2014,34(4):21-26.
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[15]王旭,侯华伟,司玲玉,等.漆酶对纤维素酶催化作用的影响[J].河南农业大学学报,2018,52(2):244-248.
[16] CARROLL A A,SOMERVILLE C C. Cellulosic biofuels[J]. Annual Review of Plant Biology,2008,60:165-182.
[17]杨天龙,王淑玲,顾招兵,等.独龙牛瘤胃细菌纤维素酶基因克隆[J].南方农业学报,2017,48(5):901-906.
[18] CHEN R. A paradigm shift in biomass technology from complete to partial cellulose hydrolysis:lessons learned from nature[J]. Bioengineered,2015,6(2):69-72.
[19] PARISUTHAM V,CHANDRAN S P,MUKHOPADHYAY A,et al. Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries[J]. Bioresource Technology,2017,239:496-506.
[20] GALAZKA J M,JIN Y S,KIM H J,et al. Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters[J]. Metabolic Engineering,2013,15:134-143.
[21] HA S J,GALAZKA J M,KIM S R,et al. Engineered saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation[J]. Proceedings of the National Academy of Sciences,2011,108(2):504-509.
[22] KIM S K,HIMMEL M E,BOMBLE Y J,et al. Expression of a cellobiose phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii improves the phosphorolytic pathway and results in a dramatic increase in cellulolytic activity[J]. Applied and Environmental Microbiology,2018,84(3):e02348.
[2] SHETH K,ALEXANDER J K. Purification and properties of beta-1,4-oligoglucan:orthophosphate glucosyltransferase from Clostridium thermocellum[J]. Journal of Biological Chemistry,1969,244(2):457-464.
[3] SASAKI T,TANAKA T,NAKAGAWA S,et al. Purification and properties of Cellvibrio gilvus cellobiose phosphorylase[J]. Biochemical Journal,1983,209(3):803-807.
[4] PUCHART V. Glycoside phosphorylases:structure,catalytic properties and biotechnological potential[J].Biotechnology Advances,2015,33(2):261-276.
[5] O’NEILL E C,PERGOLIZZI G,STEVENSON C E M,et al. Cellodextrin phosphorylase from Ruminiclostridium thermocellum:X-ray crystal structure and substrate specificity analysis[J]. Carbohydrate Research,2017,451:118-132.
[6] KUK A C,MASHALIDIS E H,LEE S Y. Crystal structure of the MOP flippase MurJ in an inward-facing conformation[J]. Nature Structural&Molecular Biology,2017,24(2):171-176.
[7] NESBΦC L,BAPTESTE E,CURTIS B,et al. The genome of Thermosipho africanus TCF52B:lateral genetic connections to the firmicutes and archaea[J]. Journal of Bacteriology,2009,191(6):1974-1978.
[8] GEFAND D H,LAWRENCE G I,REICHERT F L.Purified thermostable nucleic acid polymerase enzyme from Thermosipho africanus,US:5968799[P]. 1999-10-19.
[9] DAVIS M,SZASZ J. Thermostable alkaline phosphatase from Thermosipho africanus,US:5633138[P]. 1997-5-27.
[10] WU Y,MAO G,FAN H,et al. Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity[J]. Scientific Reports,2017,7:4849.
[11] KELLEY L A,MEZULIS S,YATES C M,et al. The phyre2 web portal for protein modeling,prediction and analysis[J]. Nature Protocols,2015,10(6):845-858.
[12]许柏英,张甜甜.铜绿微囊藻气囊结构蛋白Gvp C的表达纯化与晶体生长研究[J].山东大学学报(理学版),2018,53(11):1-8.
[13]王海波,叶雨佳,孟照辉.人甲状腺激素受体相互作用蛋白15的初步结晶实验及其在人组织表达的研究[J].中国生物工程杂志,2014,34(4):21-26.
[14] OTWINOWSKI Z,MINOR W. Processing of X-ray diffraction data collected in oscillation mode[J]. Methods in Enzymology. 1997,276:307-326.
[15]王旭,侯华伟,司玲玉,等.漆酶对纤维素酶催化作用的影响[J].河南农业大学学报,2018,52(2):244-248.
[16] CARROLL A A,SOMERVILLE C C. Cellulosic biofuels[J]. Annual Review of Plant Biology,2008,60:165-182.
[17]杨天龙,王淑玲,顾招兵,等.独龙牛瘤胃细菌纤维素酶基因克隆[J].南方农业学报,2017,48(5):901-906.
[18] CHEN R. A paradigm shift in biomass technology from complete to partial cellulose hydrolysis:lessons learned from nature[J]. Bioengineered,2015,6(2):69-72.
[19] PARISUTHAM V,CHANDRAN S P,MUKHOPADHYAY A,et al. Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries[J]. Bioresource Technology,2017,239:496-506.
[20] GALAZKA J M,JIN Y S,KIM H J,et al. Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters[J]. Metabolic Engineering,2013,15:134-143.
[21] HA S J,GALAZKA J M,KIM S R,et al. Engineered saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation[J]. Proceedings of the National Academy of Sciences,2011,108(2):504-509.
[22] KIM S K,HIMMEL M E,BOMBLE Y J,et al. Expression of a cellobiose phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii improves the phosphorolytic pathway and results in a dramatic increase in cellulolytic activity[J]. Applied and Environmental Microbiology,2018,84(3):e02348.