title: Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop.
abstract: Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable β-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k                         (cat) and K                         (m) values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application.
authors.affiliation: Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan.
authors.forename: Ya-Shan
authors.initials: YS
authors.lastname: Cheng
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.forename: Jian-Wen
authors.initials: JW
authors.lastname: Huang
authors.forename: Tzu-Hui
authors.initials: TH
authors.lastname: Wu
authors.forename: Cheng-Yen
authors.initials: CY
authors.lastname: Lin
authors.forename: Wenhua
authors.initials: W
authors.lastname: Luo
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
authors.forename: Chun-Hsiang
authors.initials: CH
authors.lastname: Huang
authors.forename: Andrew H-J
authors.initials: AH
authors.lastname: Wang
authors.forename: Je-Ruei
authors.initials: JR
authors.lastname: Liu
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
mesh_terms: D019943:Amino Acid Substitution; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D007700:Kinetics; D060847:Metabolic Engineering / Q000379:methods; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
chemical_list: D050505:Mutant Proteins; D002480:Cellulase