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Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

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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.

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Acknowledgments

We thank the National Synchrotron Radiation Research Center of Taiwan and the Shanghai Synchrotron Radiation Facility of China for beam-time allocations and data-collection assistance. This work was supported by grants from the National Science Council of Taiwan (NSC 98-2313-B002-033-MY3 to JRL), the National Basic Research Program of China (2011CB710800 to RTG), and the Tianjin Municipal Science and Technology Commission (10ZCKFSY06000 to RTG).

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Authors and Affiliations

  1. Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan

    Ya-Shan Cheng, Tzu-Hui Wu & Je-Ruei Liu

  2. Institute of Biological Chemistry, Academia Sinica, Taipei, 115, Taiwan

    Tzu-Ping Ko & Andrew H.-J. Wang

  3. Genozyme Biotechnology Inc., Taipei, 114, Taiwan

    Jian-Wen Huang & Cheng-Yen Lin

  4. AsiaPac Biotechnology Co., Ltd., Dongguan, 523808, China

    Jian-Wen Huang & Cheng-Yen Lin

  5. College of Food Science, South China Agricultural University, Guangzhou, 510642, China

    Wenhua Luo

  6. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

    Qian Li, Yanhe Ma, Chun-Hsiang Huang & Rey-Ting Guo

  7. Department of Animal Science and Technology, National Taiwan University, Taipei, 106, Taiwan

    Je-Ruei Liu

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  1. Ya-Shan Cheng
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Correspondence to Je-Ruei Liu or Rey-Ting Guo.

Additional information

Y.-S. Cheng and T.-P. Ko contributed equally to this work.

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Supp. Movie S1

TmCel12A catalysis in motion. Interestingly, all bound cellobiose molecules in the Y61G crystal, which occupied the −2 and −1 subsites, had the β-anomeric configuration at the reducing end, where the bond cleavage should have taken place, and they should represent the product-like structure. On the other hand, the bound −1 sugar in the previous E134C mutant structures had the α-anomeric configuration, and they should represent the intermediate-like structure. Together with the structure of wild-type TmCel12A–cellotetraose complex, which represents an uncleaved substrate, a composite illustration of the catalytic process is shown here. It is worth noting that every model in this movie actually comes from one of the crystal structures. In other words, they are all real observations. The protein molecules are shown as green worm models and the bound ligand as stick models with white carbon atoms and red oxygen atoms. The movie was produced by using the program PyMOL and the PDB entries 3AMH, 3AMM, 3AMN, 3AMP, 3AMQ, and 3VHN. Five subsites were observed in the substrate-binding cleft of TmCel12A. They are numbered −3, −2, −1, +1, and +2 from the non-reducing end. The double-displacement cleavage occurs between the −1 sugar and the +1 sugar. The substrate is represented by the bound cellotetraose to the wild-type enzyme. After the first nucleophilic attack by Glu134, an intermediate with the α-anomeric configuration in the −1 sugar is produced, and it is represented by several complex structures of the E134C mutant. The cleaved moiety at the reducing end, originally bound to the +1 and +2 sites, departs from the enzyme. Then the second nucleophilic attack by a water molecule takes place, reversing the α-anomer to a β-anomer at the −1 sugar. The product at the non-reducing end is eventually replaced by a new substrate molecule, and another cycle of the catalytic reaction begins. (MOV 906 kb)

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Cheng, YS., Ko, TP., Huang, JW. et al. Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop. Appl Microbiol Biotechnol 95, 661–669 (2012). https://doi.org/10.1007/s00253-011-3791-4

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