耐热普鲁兰酶CBM68结构域中关键位点对其酶学性质的影响
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摘要
通过对Anoxybacillus sp.LM18-11来源的耐热普鲁兰酶(Pul A)与麦芽三糖共结晶的晶体结构分析,在新命名的底物结合域CBM68中预测出4个与底物结合有关的氨基酸位点(Y14、D16、K62和R96),分别将4个氨基酸位点突变为丙氨酸,得到突变体Pul A~(Y14A)、Pul A~(D16A)、Pul A~(K62A)和Pul A~(R96A)。其中,突变酶Pul A~(Y14A)和Pul A~(R96A)的底物结合力及催化效率与野生酶Pul A相比均有明显降低,其K_m值分别比Pul A提高了4.2倍和2.5倍,k_(cat)/K_m分别为Pul A的29%和37%。同时,Pul A~(Y14A)和Pul A~(R96A)的最适作用温度均降低了10℃,Pul A~(R96A)的最适作用p H也降低了0.5个单位。说明Y14和R96是CBM68结构域中的关键氨基酸位点,这两个位点的发现为进一步探究CBM68结构域的作用机制奠定了基础。
Based on the analysis of the crystalline structure of pullulananse( Pul A)-maltotetraose complex,four key amino acid sites( Y14,D16,K62 and R96) in a new carbohydrate-binding module CBM68 closely related to substrate binding were discovered. The thermostable pullulanase( Pul A) was derived from Anoxybacillus sp. LM18-11. Four mutants of Pul A~(Y14A),Pul A~(D16A),Pul A~(K62) Aand Pul A~(R96A)were obtained respectively by site-directed mutation.Among them,Pul A~(Y14A)and Pul A~(R96A)showed lower affinity for substrate and lower catalytic efficiency compared with the wild-type pullulanase( Pul A). The K_mvalues of Pul A~(Y14A)and Pul A~(R96A)were 4. 2 times and 2. 5 times higher than that of Pul A,respectively. The k_(cat)/ K_mvalues of Pul A~(Y14A)and Pul A~(R96A)only remained 29% and 37% of the original k_(cat)/ K_mvalue( Pul A),respectively. Moreover,the optimum temperatures for both Pul A~(Y14A)and Pul A~(R96A)were 10 ℃lower than that for the wild-type pullulanase( Pul A). And Pul A~(R96A)also showed decreasing of the optimum p H by 0. 5units. All the results in this work have proved the significance of the key amino acid sites Y14 and R96 on the structure CBM68. Further study on the function of the two key amino acid sites could enhance the understanding of the functional mechanism of CBM68.
Based on the analysis of the crystalline structure of pullulananse( Pul A)-maltotetraose complex,four key amino acid sites( Y14,D16,K62 and R96) in a new carbohydrate-binding module CBM68 closely related to substrate binding were discovered. The thermostable pullulanase( Pul A) was derived from Anoxybacillus sp. LM18-11. Four mutants of Pul A~(Y14A),Pul A~(D16A),Pul A~(K62) Aand Pul A~(R96A)were obtained respectively by site-directed mutation.Among them,Pul A~(Y14A)and Pul A~(R96A)showed lower affinity for substrate and lower catalytic efficiency compared with the wild-type pullulanase( Pul A). The K_mvalues of Pul A~(Y14A)and Pul A~(R96A)were 4. 2 times and 2. 5 times higher than that of Pul A,respectively. The k_(cat)/ K_mvalues of Pul A~(Y14A)and Pul A~(R96A)only remained 29% and 37% of the original k_(cat)/ K_mvalue( Pul A),respectively. Moreover,the optimum temperatures for both Pul A~(Y14A)and Pul A~(R96A)were 10 ℃lower than that for the wild-type pullulanase( Pul A). And Pul A~(R96A)also showed decreasing of the optimum p H by 0. 5units. All the results in this work have proved the significance of the key amino acid sites Y14 and R96 on the structure CBM68. Further study on the function of the two key amino acid sites could enhance the understanding of the functional mechanism of CBM68.
引文
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[12]XU Jiang-yong,REN Fei-fei,HUANG Chun-hsiang,et al.Functional and structural studies of pullulanase from Anoxybacillus sp.LM18-11[J].Proteins,2013,82(9):1685-1 693.
[13]李红,孙红军.国内外普鲁兰酶市场分析及预测报告(2003、2004年度)[R].北京:理德斯普企业管理咨询有限公司,2004.
[14]ZHOU Cheng,XUE Yan-feng,ZHANG Yue-ling,et al.Recombinant expression and characterization of Thermoanaerobacter tengcongensis MB4 thermostableα-glucosidase with regioselectivity for high yield isomaltooligosaccharides synthesis[J].Journal of Microbiology and Bchnology,2009,19(12):1 547-1 556.
[15]CHEN C,XUE Yan-fen,MA Yan-he.Enhancing the thermostability ofα-glucosidase from Thermoanaerobacter tengcongensis MB4 by single proline substitution[J].Bioscience and Bioengineering,2010,110(1):12-17.
[16]GUILLEN D,S SANCHEZ,RODRIGUEZ-SANOJA R.Carbohydrate-binding domains:multiplicity of biological roles[J].Appl Microbiol Biotechnol,2010,85(5):1 241-1 249.
[2]姜楠,宋诙,王萍.普鲁兰酶及其分泌相关蛋白的研究进展[J].微生物学报,2011,51(6):725-731.
[3]SIEW Ling Hii,JOO Shun TAN,TAU Chuan Ling,et al.Pullulanase:Role in starch hydrolysis and potential industrial applications[J].Enzyme Res,2012,2012:921 362.
[4]BORASTON A B,HEALEY M,KLASSEN J,et al.A structural and functional analysis of alpha-glucan recognition by family 25 and 26 carbohydrate-binding modules reveals a conserved mode of starch recognition[J].Biol Chem,2006,281(1):587-98.
[5]MIKAMI B,IWAMOTO H,MALLE D,et al.Crystal structure of pullulanase:evidence for parallel binding of oligosaccharides in the active site[J].Mol Biol,2006,359(3):690-707.
[6]TURKENBURG J P,BRZOZOWSKI A M,SVENDSEN A,et al.Structure of a pullulanase from Bacillus acidopullulyticus[J].Proteins,2009,76(2):516-519.
[7]VENDITTO I,NAJMUDIN S,Lus A S,et al.Family 46carbohydrate-binding modules contribute to the enzymatic hydrolysis of xyloglucan andβ-1,3-1,4-Glucans through distinct mechanisms[J].Journal of Biological Chemistry,2015,290(17):10 572-10 586.
[8]STROBEL K L,PFEIFFER K A,HARVEY W B,et al.Structural insights into the affinity of Cel7A carbohydratebinding module for lignin[J].Journal of Biological Chemistry,2015,290(37):22 818-22 826.
[9]HARA M,SUGIMOTO H,UEMURA M,et al.Involvement of Gln679,in addition to Trp687,in chitin-binding activity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12[J].Journal of Biochemistry,2013,154(2):185-193.
[10]DUAN Xu-guo,CHEN Jian,WU Jing.Improving the thermostability and catalytic efficiency of Bacillus deramificans pullulanase by site-directed mutagenesis[J].Applied and Environmental Microbiology,2013,79(13):4 072-4 077.
[11]甄杰,胡政,李树芳,等.一个新型耐热普鲁兰酶的结构与功能[J].生物工程学报,2014,30(1):119-128.
[12]XU Jiang-yong,REN Fei-fei,HUANG Chun-hsiang,et al.Functional and structural studies of pullulanase from Anoxybacillus sp.LM18-11[J].Proteins,2013,82(9):1685-1 693.
[13]李红,孙红军.国内外普鲁兰酶市场分析及预测报告(2003、2004年度)[R].北京:理德斯普企业管理咨询有限公司,2004.
[14]ZHOU Cheng,XUE Yan-feng,ZHANG Yue-ling,et al.Recombinant expression and characterization of Thermoanaerobacter tengcongensis MB4 thermostableα-glucosidase with regioselectivity for high yield isomaltooligosaccharides synthesis[J].Journal of Microbiology and Bchnology,2009,19(12):1 547-1 556.
[15]CHEN C,XUE Yan-fen,MA Yan-he.Enhancing the thermostability ofα-glucosidase from Thermoanaerobacter tengcongensis MB4 by single proline substitution[J].Bioscience and Bioengineering,2010,110(1):12-17.
[16]GUILLEN D,S SANCHEZ,RODRIGUEZ-SANOJA R.Carbohydrate-binding domains:multiplicity of biological roles[J].Appl Microbiol Biotechnol,2010,85(5):1 241-1 249.