Modulating the function of a β-1,3-glucanosyltransferase  to that of an endo-β-1,3-glucanase by structure-based protein  engineering.
A glycoside hydrolase (GH) family 17 β-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other β-1,3-glucanases,despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH family 17 β-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a β-1,3-glucanase. Mutant E158A exhibited high hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold higher than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-β-1,3-glucanase. Our study provides novel insight into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.
College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, No.17 Qinghua Donglu, Haidian District, Post Box 294, Beijing, 100083, China.
Zhen
Z
Qin
Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, No.17 Qinghua Donglu, Haidian District, Post Box 294, Beijing, 100083, China. yanqj@cau.edu.cn.
Qiaojuan
Q
Yan
College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, No.17 Qinghua Donglu, Haidian District, Post Box 294, Beijing, 100083, China.
Shaoqing
S
Yang
College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, No.17 Qinghua Donglu, Haidian District, Post Box 294, Beijing, 100083, China. zhqjiang@cau.edu.cn.
Zhengqiang
Z
Jiang
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002855:Chromatography, Thin Layer; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D020103:Rhizomucor / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
C117939:1,3-beta-glucanosyltransferase; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase