生物信息学分析葡聚糖蔗糖酶的结构和功能特性

doi:10.11924/j.issn.1000-6850.casb2023-0670

中国农学通报 ›› 2024, Vol. 40 ›› Issue (18): 105-114.doi: 10.11924/j.issn.1000-6850.casb2023-0670

生物信息学分析葡聚糖蔗糖酶的结构和功能特性

蔡华扬¹(), 于连升¹, 李腾鑫¹, 林宜萌¹^,², 杜仁鹏¹^,²()

¹ 黑龙江大学，生命科学学院，农业微生物技术教育部工程研究中心，黑龙江省寒区植物基因与生物发酵重点实验室，黑龙江省普通高校微生物重点实验室，哈尔滨 150080
² 河北环境工程学院，河北省农业生态安全重点实验室，河北秦皇岛 066102

收稿日期:2023-09-20 修回日期:2024-01-15 出版日期:2024-06-25 发布日期:2024-06-18
通讯作者:
杜仁鹏，男，1989年出生，黑龙江大兴安岭人，副教授，博士，研究方向：微生物次生代谢产物与生理。通信地址：150080 黑龙江哈尔滨南岗区学府路74号生命科学学院，Tel：0451-86609134，E-mail：durenpeng20096292@163.com。
作者简介:
蔡华扬，男，2002年出生，广东惠州人，本科生，研究方向：乳酸菌胞外多糖生物合成机制。通信地址：150080 黑龙江哈尔滨市南岗区学府路74号，Tel：0451-86609134，E-mail：1667855847@qq.com
基金资助:
黑龙江省自然科学基金优秀青年基金项目“乳酸明串珠菌群体感应和第二信使介导右旋糖酐合成的信号通路研究”(YQ2021C030); 中国博士后科学基金资助项目“c-di-GMP和c-di-AMP介导乳酸明串珠菌L2胞外多糖合成的分子调控机制研究”(2022MD713755); 黑龙江省博士后资助项目“柠檬明串珠菌胞外多糖合成途径中信号网络的作用机制研究”(LBH-Z21082); 新时代龙江优秀硕士、博士学位论文项目“肠膜明串珠菌DRP105右旋糖酐生物合成机制的研究”(LJYXL2022-020); 河北省农业生态安全重点实验室开放基金课题项目“乳酸菌胞外多糖的分离纯化及构效关系研究”(2023SYSJJ17); 黑龙江省省属高等学校基本科研业务费科研项目“c-di-AMP调控肠膜明串珠菌DRP105胞外多糖的生物合成机理”(2022-KYYWF-1075)

Analysis of Structure and Functional Properties of Glucansucrase Based on Bioinformatics Method

CAI Huayang¹(), YU Liansheng¹, LI Tengxin¹, LIN Yimeng¹^,², Du Renpeng¹^,²()

¹ Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education/ Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region/ Key Laboratory of Microbiology, College of Heilongjiang Province/ School of Life Sciences, Heilongjiang University, Harbin 150080
² Hebei Key Laboratory of Agroecological Safety, Hebei University of Environmental Engineering, Qinhuangdao, Hebei 066102

Received:2023-09-20 Revised:2024-01-15 Published:2024-06-25 Online:2024-06-18

摘要/Abstract

摘要：

葡聚糖蔗糖酶是一种重要的糖基转移酶(Glucosyltransferase, GTF)，对于乳酸菌合成胞外多糖具有关键作用，能够以蔗糖为底物合成葡聚糖或低聚糖，是乳酸菌(Lactic acid bacteria，LAB)合成胞外多糖(Exopolysaccharide，EPS)的关键酶蛋白。然而乳酸菌代谢系统复杂，EPS生物合成机制未得到全面解析，限制了EPS的应用。为了进一步解析乳酸菌EPS的生物合成机制，表征不同来源葡聚糖蔗糖酶的结构，探明酶的催化调控机制是必要手段。本研究以肠膜明串珠菌(Leuconostoc mesenteroides) DRP105的葡聚糖蔗糖酶gtfB基因为目标序列，利用生物信息学技术对葡聚糖蔗糖酶GtfB结构和功能进行了预测分析。研究结果显示：GtfB属于GH70家族，是一种胞外酶，不存在跨膜区，含有7个保守区域和39条重复序列。其理论分子量为308986.21，理论等电点为4.59，属于酸性蛋白质。磷酸化位点含110个Thr、89个Ser、53个Tyr，GtfB含有5个结构域，呈U型折叠，活性中心在A结构域，含有(β/α)₈桶状结构。分子对接预测分析表明，蔗糖与GtfB的活性口袋结合紧密。这些结果初步探明了GtfB具有葡聚糖蔗糖酶的特征结构和功能，证明其在乳酸菌EPS合成途径中的关键调控作用，为其在EPS生产过程中的应用提供了理论基础。

关键词: 肠膜明串珠菌, 葡聚糖蔗糖酶, 生物信息学分析, 结构预测, 分子对接

Abstract:

Glucansucrase is a glycosyltransferase (GTF) that can synthesize dextran or oligosaccharides with sucrose as a substrate, and is a key enzyme protein for the synthesis of exopolysaccharide (EPS) by lactic acid bacteria (LAB). However, the metabolic system of LAB is complex, and the mechanism of EPS biosynthesis has not been elucidated, which limits the development of EPS. In order to further analyze the biosynthetic mechanism of LAB EPS and characterize the structure of dextransucrase from different sources, it is necessary to explore the catalytic regulation mechanism of enzyme. In this study, the structure and function of glucansucrase GtfB from Leuconostoc mesenteroides DRP105 were predicted with bioinformatics techniques. The results showed that GtfB belonged to the GH70 family, which was an extracellular enzyme with no transmembrane region, containing 7 conserved regions and 39 repeats. The theoretical molecular weight of GtfB was 308986.21, and the theoretical isoelectric point was 4.59, which was an acidic protein. The phosphorylation site contained 110 Thr, 89 Ser, and 53 Tyr, and GtfB contained 5 domains, which were U-folded, and the active center was in the A domain, containing (β/α)₈ barrel-like structures. Molecular docking prediction analysis showed that sucrose was tightly bound to the active pocket of GtfB. The results of this study preliminarily revealed the characteristic structure and function of GtfB as a glucanase, proving that this enzyme is a key regulatory enzyme in the EPS synthesis pathway of LAB and has potential application value in the EPS production process.

Key words: Leuconostoc mesenteroides, glucansucrase, bioinformatics analysis, structure prediction, molecular docking

蔡华扬, 于连升, 李腾鑫, 林宜萌, 杜仁鹏. 生物信息学分析葡聚糖蔗糖酶的结构和功能特性[J]. 中国农学通报, 2024, 40(18): 105-114.

CAI Huayang, YU Liansheng, LI Tengxin, LIN Yimeng, Du Renpeng. Analysis of Structure and Functional Properties of Glucansucrase Based on Bioinformatics Method[J]. Chinese Agricultural Science Bulletin, 2024, 40(18): 105-114.

图/表 10

图1. gtfB开放阅读框分析

图2. GtfB多序列比对结果图

图3. GtfB的保守序列分析

图4. GtfB蛋白进化树分析

氨基酸极性	氨基酸	数量	比例/%
酸性氨基酸	Glu (E)	108	3.9
酸性氨基酸	Asp (D)	256	9.2
碱性氨基酸	Arg (R)	54	1.9
	Lys (K)	174	6.2
	His (H)	34	1.2
非极性氨基酸	Ala (A)	206	8.3
	Val (V)	175	6.3
	Leu (L)	188	6.7
	Ile (I)	126	4.5
	Trp (W)	42	1.5
	Met (M)	52	1.9
	Phe (F)	90	3.2
	Pro (P)	76	2.7
不带电荷极性氨基酸	Asn (N)	231	8.3
	Cys (C)	1	0
	Gln (Q)	153	5.5
	Gly (G)	227	8.1
	Ser (S)	193	6.9
	Thr (T)	250	8.9
	Tyr (Y)	161	5.8

表1. GtfB氨基酸组成

图5. 蛋白质理化性质分析 (a)磷酸化位点分析；(b)疏水性分析

图6. GtfB蛋白质二级结构预测

图7. 同源建模法预测GtfB的三维结构 (a)预测质量评估报告，(b)GtfB三维结构图

图8. GtfB与蔗糖分子对接预测

图9. GtfB蛋白互作网络

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生物信息学分析葡聚糖蔗糖酶的结构和功能特性

Analysis of Structure and Functional Properties of Glucansucrase Based on Bioinformatics Method

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