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Disulfide bridges as essential elements for the thermostability of lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor

Magali Tanghe (UGent) , Barbara Danneels (UGent) , Matthias Last, Koen Beerens (UGent) , Ingeborg Stals (UGent) and Tom Desmet (UGent)
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Abstract
Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80 degrees C while its apparent melting temperature (T-m) is only 51 degrees C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in T-m, ranging from 2 to 9 degrees C. Combining the positive mutations yielded additional improvements (up to 19 degrees C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12 degrees C increase in T-m and was able to retain and was able to retain no less than 60% of its activity after heat treatment.
Keywords
disulfide bridge, lytic polysaccharide monooxygenases, Streptomyces coelicolor LPMO10C, thermal stability, LIGNOCELLULOSIC BIOMASS HYDROLYSIS, SACCHAROMYCES-CEREVISIAE, SATURATION MUTAGENESIS, THERMAL-STABILITY, PROTEIN, CELLULOSE, BONDS, EXPRESSION, DEGRADATION, CYSTEINE

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Chicago
Tanghe, Magali, Barbara Danneels, Matthias Last, Koen Beerens, Ingeborg Stals, and Tom Desmet. 2017. “Disulfide Bridges as Essential Elements for the Thermostability of Lytic Polysaccharide Monooxygenase LPMO10C from Streptomyces Coelicolor.” Protein Engineering Design & Selection 30 (5): 401–408.
APA
Tanghe, M., Danneels, B., Last, M., Beerens, K., Stals, I., & Desmet, T. (2017). Disulfide bridges as essential elements for the thermostability of lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor. PROTEIN ENGINEERING DESIGN & SELECTION, 30(5), 401–408.
Vancouver
1.
Tanghe M, Danneels B, Last M, Beerens K, Stals I, Desmet T. Disulfide bridges as essential elements for the thermostability of lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor. PROTEIN ENGINEERING DESIGN & SELECTION. 2017;30(5):401–8.
MLA
Tanghe, Magali et al. “Disulfide Bridges as Essential Elements for the Thermostability of Lytic Polysaccharide Monooxygenase LPMO10C from Streptomyces Coelicolor.” PROTEIN ENGINEERING DESIGN & SELECTION 30.5 (2017): 401–408. Print.
@article{8522484,
  abstract     = {Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80 degrees C while its apparent melting temperature (T-m) is only 51 degrees C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in T-m, ranging from 2 to 9 degrees C. Combining the positive mutations yielded additional improvements (up to 19 degrees C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12 degrees C increase in T-m and was able to retain and was able to retain no less than 60% of its activity after heat treatment.},
  author       = {Tanghe, Magali and Danneels, Barbara and Last, Matthias and Beerens, Koen and Stals, Ingeborg and Desmet, Tom},
  issn         = {1741-0126},
  journal      = {PROTEIN ENGINEERING DESIGN & SELECTION},
  keywords     = {disulfide bridge,lytic polysaccharide monooxygenases,Streptomyces coelicolor LPMO10C,thermal stability,LIGNOCELLULOSIC BIOMASS HYDROLYSIS,SACCHAROMYCES-CEREVISIAE,SATURATION MUTAGENESIS,THERMAL-STABILITY,PROTEIN,CELLULOSE,BONDS,EXPRESSION,DEGRADATION,CYSTEINE},
  language     = {eng},
  number       = {5},
  pages        = {401--408},
  title        = {Disulfide bridges as essential elements for the thermostability of lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor},
  url          = {http://dx.doi.org/10.1093/protein/gzx014},
  volume       = {30},
  year         = {2017},
}

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