title: Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency.
abstract: Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases usingNfEG12A fromNeosartorya fischeriP1 and EG (PDB 1KS4) fromAspergillus nigerAcidophilic and thermophilicNfEG12A had the highest catalytic efficiency (kcat/Km, 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced intoNfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (kcat/Km) of these enzymes (in descending order,NfEG12A [100%],NfEG12A-FN [140%], andNfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (kcat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCELoop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the -1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases,NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (kcat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Hong
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.|College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, People's Republic of China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xia
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.|College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, People's Republic of China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Huifang
authors.initials: H
authors.lastname: Cao
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Ma
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China baiyingguo@caas.cn binyao@caas.cn.
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China baiyingguo@caas.cn binyao@caas.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D008031:Lignin / Q000378:metabolism*; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D055326:Neosartorya / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains / Q000235:genetics*; D013379:Substrate Specificity; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D005936:Glucans; D047071:beta-Glucans; C036909:lignocellulose; D008031:Lignin; D006026:Glycoside Hydrolases; D002480:Cellulase; C015968:lichenin
keywords: GH12 β-endoglucanase; Neosartorya fischeri; catalytic efficiency; hydrogen bond; loop