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

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

Abstract

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

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.

Figures/Tables 10

References 39

[1]	GAO Z, WU C, WU J, et al. Antioxidant and anti-inflammatory properties of an aminoglycan-rich exopolysaccharide from the submerged fermentation of Bacillus thuringiensis[J]. International journal of biological macromolecules, 2022, 220:1010-1120.
[2]	KIM I, CHHETRI G, SO Y, et al. Characteristics and biological activity of exopolysaccharide produced by Lysobacter sp. MMG2 isolated from the roots of Tagetes patula[J]. Microorganisms, 2022, 10(7):1257.
[3]	FARAG M M S, MOGHANNEM S A M, SHEHABELDINE A M, et al. Antitumor effect of exopolysaccharide produced by Bacillus mycoides[J]. Microbial pathogenesis, 2020,140:103947.
[4]	ADEBAYO-TAYO B, FASHOGBON R. In vitro antioxidant, antibacterial, in vivo immunomodulatory, antitumor and hematological potential of exopolysaccharide produced by wild type and mutant Lactobacillus delbureckii subsp. bulgaricus[J]. Heliyon, 2020, 6(2):e03268.
[5]	ZHANG Q, WANG J, SUN Q, et al. Characterization and Antioxidant activity of released exopolysaccharide from potential probiotic Leuconostoc mesenteroides LM187[J]. Journal of microbiology and biotechnology, 2021, 31(8):1144-1153.
[6]	李洋. 肠膜明串珠菌SN-8胞外多糖分离纯化, 结构鉴定及功能特性研究[D]. 沈阳: 沈阳农业大学, 2020.
[7]	LJUBIC V, PERENDIJA J, CVETKOVIC S, et al. Removal of Ni²⁺ ions from contaminated water by new exopolysaccharide extracted from K. oxytoca J7 as Biosorbent[J]. Journal of polymers and the environment, 2023,21:s10924.
[8]	WU J, HAN X, YE M, et al. Exopolysaccharides synthesized by lactic acid bacteria:biosynthesis pathway, structure-function relationship, structural modification and applicability[J]. Critical reviews in food science and nutrition, 2022:1-22.
[9]	CHEN Z, NI D, ZHANG W, et al. Lactic acid bacteria-derived α-glucans: from enzymatic synthesis to miscellaneous applications[J]. Biotechnology advances, 2021,47:107708.
[10]	SHI K, AN W, MENG Q, et al. Partial characterization and lyoprotective activity of exopolysaccharide from Oenococcus oeni 28A-1[J]. Process biochemistry, 2021, 101:128-136.
[11]	MIYAMOTO J, SHIMIZU H, HISA K, et al. Host metabolic benefits of prebiotic exopolysaccharides produced by Leuconostoc mesenteroides[J]. Gut microbes, 2023, 15(1):2161271.
[12]	王小杰, 张华, 陈其欣, 等. 肠膜明串珠菌产葡聚糖与低聚糖及其产物应用研究进展[J]. 中国乳品工业, 2019, 47(6):33-36.
[13]	GANGOITI J, PIJNING T, DIJKHUIZEN L. Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose[J]. Biotechnology advances, 2018, 36(1):196-207. doi: S0734-9750(17)30129-5 pmid: 29133008
[14]	DU R P, ZHOU Z J, HAN Y. Functional identification of the dextransucrase gene of Leuconostoc mesenteroidesDRP105[J]. International journal of molecular sciences, 2020, 21(18):6596.
[15]	YAN M H, WANG B H, XU X F, et al. Molecular and functional study of a branching sucrase-like glucansucrase reveals an evolutionary intermediate between two subfamilies of the GH70 enzymes[J]. Applied and environmental microbiology, 2018, 84(9):e02810-e02817.
[16]	ARGUELLO-MORALES M A, REMAUD-SIMEON M, PIZZUT S, et al. Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355[J]. FEMS microbiology letters, 2000, 182(1):81-85.
[17]	PIJNING T, VUJICIC-ZAGAR A, KRALJ S, et al. Structure of the alpha-1,6/alpha-1,4-specific glucansucrase GTFA from Lactobacillus reuteri 121[J]. Acta crystallographica section f-structural biology communications, 2012,68:1448-1454.
[18]	张皓, 祖航天, 胡杰, 等. 一种产碱性葡聚糖蔗糖酶菌株的筛选、鉴定及其合成低聚糖的研究[J]. 食品科技, 2021, 46(7):22-28.
[19]	袁宇, 蓝艳禹, 黄臣, 等. 响应面法优化肠膜明串珠菌生物合成右旋糖酐工艺条件[J]. 食品研究与开发, 2018, 39(7):187-192.
[20]	叶广彬, 陈源红, 王长丽, 等. 柠檬明串珠菌TD1产胞外多糖条件的响应面法优化及其抗氧化性研究[J]. 中国酿造, 2018, 37(11):70-75. doi: 10.11882/j.issn.0254-5071.2018.11.015
[21]	LASKOWSKI R A, SWINDELLS M B. LigPlot+:multiple ligand-protein interaction diagrams for drug discovery[J]. Journal of chemical nfiormation and modeling, 2011, 51(10):2778-2786.
[22]	SHAH D S H, JOUCLA G, REMAUD-SIMEON M, et al. Conserved repeat motifs and glucan binding by glucansucrases of Oral Streptococci and Leuconostoc mesenteroides[J]. Journal of bacteriology, 2004, 186(24):8301-8308.
[23]	WANGPAIBOON K, PITAKCHATWONG C, PANPETCH P, et al. Modified properties of alternan polymers arising from deletion of SH3-like motifs in Leuconostoc citreum ABK-1 alternansucrase[J]. Carbohydrate polymers, 2019,220:103-109.
[24]	MENG X, GANGOITI J, DE KOK N, et al. Biochemical characterization of two GH70 family 4,6-α-glucanotransferases with distinct product specificity from Lactobacillus aviarius subsp. aviarius DSM 20655[J]. Food chemistry, 2018,253:236-246.
[25]	JOHNSON J L, YARON T M, HUNTSMAN E M, et al. An atlas of substrate specificities for the human serine/threonine kinome[J]. Nature, 2023, 613(7945):759.
[26]	MACZUGA N, TRAN E N H, MORONA R. Subcellular localization of the enterobacterial common antigen GT-E-like glycosyltransferase, WecG[J]. Molecular microbiology, 2022, 118(4):403-416.
[27]	刘娜, 田雨菁, 冯哲瀚, 等. 蓝莓3-O-葡萄糖基转移酶基因克隆及生物信息学分析[J]. 华北农学报, 2023, 38(2):120-128. doi: 10.7668/hbnxb.20193980
[28]	陈琦, 李春秀, 郑高伟, 等. 工业蛋白质构效关系的计算生物学解析[J]. 生物工程学报, 2019, 35(10):1829-1842.
[29]	YU L, QIAN Z, GE J, et al. Glucansucrase produced by lactic acid bacteria: structure, properties, and applications[J]. Fermentation, 2022, 8(11):629.
[30]	VUJICIC-ZAGAR A, PIJNING T, KRALJ S, et al. Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes[J]. Proceedings of the national academy of sciences of the united states of America, 2010, 107(50):21406-21411.
[31]	MOLINA M, CIOCI G, MOULIS C, et al. Bacterial α-glucan and branching sucrases from GH70 family: discovery, structure-function relationship studies and engineering[J]. Microorganisms, 2021, 9(8):1607.
[32]	杨卫康. 4,6-α-葡聚糖转移酶的挖掘、定性及产物特异性研究[D]. 无锡: 江南大学, 2023.
[33]	谭姚姚, 何贺贺, 王宏萌, 等. Clostridium butyricum蔗糖磷酸化酶的酶学性质及其功能研究[J]. 广西科学, 2022, 29(6):1094-1102.
[34]	WANG J, ZHANG J, WANG L, et al. Continuous production of fructooligosaccharides by recycling of the thermal-stable β-fructofuranosidase produced by Aspergillus niger[J]. Biotechnology letters, 2021, 43(6):1175-1182.
[35]	RODRIGUES S, LONA L M F, FRANCO T T. Effect of phosphate concentration on the production of dextransucrase by Leuconostoc mesenteroides NRRL B512F[J]. Bioprocess and biosystems engineering, 2003, 26(1):57-62.
[36]	虞宁馨, 于连升, 齐心彤, 等. 肠膜明串珠菌葡聚糖蔗糖酶生物信息学分析[J]. 黑龙江大学自然科学学报, 2023, 65(5):1602-1606.
[37]	原晓龙, 陈剑, 陈中华, 等. 滇牡丹类黄酮7-O-葡萄糖基转移酶基因的鉴定与表达分析[J]. 西部林业科学, 2018, 47(5):19-25.
[38]	李先良, 李居宁, 李傲, 等. 棉花木葡聚糖转移/水解酶基因克隆和分析[J]. 湖北农业科学, 2017, 56(4):756-761.
[39]	杜仁鹏. 肠膜明串珠菌DRP105右旋糖酐生物合成机制的研究[D]. 天津: 天津大学, 2021.

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

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