3VHN

Y61G mutant of Cellulase 12A from thermotoga maritima

Summary for 3VHN

• Entry DOI: 10.2210/pdb3vhn/pdb
• Related: 3AMH 3AMM 3AMN 3AMP 3VHO 3VHP 3amq
• Related PRD ID: PRD_900005
• Descriptor: Endo-1,4-beta-glucanase, beta-D-glucopyranose-(1-4)-beta-D-glucopyranose (3 entities in total)
• Functional Keywords: jelly roll, hydrolase, cellulose
• Biological source: Thermotoga maritima
• Total number of polymer chains: 8
• Total formula weight: 248126.30

Authors

Cheng, Y.-S.,Ko, T.-P.,Guo, R.-T.,Liu, J.-R. (deposition date: 2011-08-30, release date: 2012-07-11, Last modification date: 2023-11-08)

Primary citation

Cheng, Y.-S.,Ko, T.-P.,Huang, J.W.,Wu, T.H.,Lin, C.Y.,Luo, W.,Li, Q.,Ma, Y.,Huang, C.H.,Wang, A.H.,Liu, J.-R.,Guo, R.-T.
Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop
Appl.Microbiol.Biotechnol., 95:661-669, 2012

PubMed 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.

PubMed: 22170108
DOI: 10.1007/s00253-011-3791-4

Experimental method

X-RAY DIFFRACTION (2.5 Å)