环糊精葡萄糖基转移酶钙离子结合位点结构与功能分析  

Structure and Function Analysis of Calcium Binding Sites in Cyclodextrin Glucanotransferase

作　　者：班宵逢[1] 李才明[1] 鲍春辉[1] 顾正彪[1,2] 李兆丰[1,2] 

机构地区：[1]江南大学食品学院,无锡214122 [2]江南大学食品科学与技术国家重点实验室,无锡214122

出　　处：《生物化学与生物物理进展》2013年第12期1239-1246,共8页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(31101228);江苏省自然科学基金(BK2011152);霍英东教育基金会高等院校青年教师基金(131069);"十二五"国家科技支撑计划(2012BAD34B07)资助项目~~

摘　　要：通过多重序列比对和晶体结构分析发现,钙离子结合位点CaⅠ和CaⅡ普遍存在于环糊精葡萄糖基转移酶(CGT酶)中,且两个位点处氨基酸残基具有较高的保守性,而钙离子结合位点CaⅢ仅存在于少数CGT酶中.此外,研究发现,钙离子结合位点可能与CGT酶的环化活力、热稳定性和产物特异性密切相关.Cyclodextrin glucanotransferase （CGTase, EC 2.4.1.19） is an extracellular enzyme capable of producing cyclodextrins through an intramolecular transglycosylation reaction. With the application of cyclodextrins expanding in the industries related to food, pharmaceuticals, cosmetic, etc, CGTase has become the focus of scientific research nowadays. Calcium binding sites widely exit in a-amylase family. Previous studies indicated that these sites had very important roles for α-amylase. It was known that CGTases also possess two or three calcium binding sites. However, their structure and function are not very clear. In the present study, structure and function of calcium binding sites in CGTases were analyzed. Sequence comparisons were performed using the ClustalX 1.8 sequence alignment program. Based on the results and crystal structure analysis, it was found that calcium binding sites CaⅠ and Ca Ⅱ exist commonly in CGTase. Most amino acids at these two calcium binding sites are highly conserved, but the residue 29 at Ca Ⅰ and residue 199 at Ca Ⅱ have significant differences between different types of CGTases. The residue 29 in α-CGTase primarily producing α-cyclodextrin or γ-CGTase primarily producing γ-cyclodextrin is Asp, while others are Asn. The residue 199 in γ-CGTase is Ser, while others are Asp. Calcium binding site Ca m only exists in few CGTases. The site consists of residues 315 and 577. In addition, site-directed mutagenesis was used to investigate the functions of calcium binding sites in CGTases. The replacement of Asp29 by Asn and Arg resulted in 23% and 35% increase in β-cyclization activity, respectively. Mutant D29R and D315A showed higher stability than wild-type CGTase at 60 ℃. Moreover, the mutant D315A had higher β- and γ-cyclodextrin specificity. These results suggested that calcium binding sites might be related to cycling activity, thermal stability, and product specificity of CGTases, which provided the directions for further revealing biological functions of calcium b

关 键 词：环糊精葡萄糖基转移酶 钙离子结合位点 结构 功能 

分 类 号：Q814[生物学—生物工程] Q71