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Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra

Zumarraga, Miren; Camarero, Susana; Shleev, Sergey LU ; Martinez-Arias, Arturo; Ballesteros, Antonio; Plou, Francisco J and Alcalde, Miguel (2008) In Proteins 71(1). p.250-260
Abstract
The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most Of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a nonmutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in... (More)
The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most Of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a nonmutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S. cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with similar to 3-fold better kinetics than parent type showed two consecutive amino acid changes (G614D -GGC/GAC- and E615K -GAG/AAG-) because of the in vivo shuffling of the mutant libraries. Both mutations are located in the C-terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C-terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C-terminal tail for the functionality of the ascomycete laccases. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
protein folding, C-terminal tail, in vivo assembly, laccase
in
Proteins
volume
71
issue
1
pages
250 - 260
publisher
John Wiley & Sons
external identifiers
  • wos:000254035500024
  • scopus:40549139786
ISSN
0887-3585
DOI
10.1002/prot.21699
language
English
LU publication?
yes
id
7ccae9bd-1615-4b8b-8c1f-4cc969d9314f (old id 1185252)
date added to LUP
2008-09-03 10:37:39
date last changed
2017-08-27 04:49:35
@article{7ccae9bd-1615-4b8b-8c1f-4cc969d9314f,
  abstract     = {The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most Of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a nonmutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S. cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with similar to 3-fold better kinetics than parent type showed two consecutive amino acid changes (G614D -GGC/GAC- and E615K -GAG/AAG-) because of the in vivo shuffling of the mutant libraries. Both mutations are located in the C-terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C-terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C-terminal tail for the functionality of the ascomycete laccases.},
  author       = {Zumarraga, Miren and Camarero, Susana and Shleev, Sergey and Martinez-Arias, Arturo and Ballesteros, Antonio and Plou, Francisco J and Alcalde, Miguel},
  issn         = {0887-3585},
  keyword      = {protein folding,C-terminal tail,in vivo assembly,laccase},
  language     = {eng},
  number       = {1},
  pages        = {250--260},
  publisher    = {John Wiley & Sons},
  series       = {Proteins},
  title        = {Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra},
  url          = {http://dx.doi.org/10.1002/prot.21699},
  volume       = {71},
  year         = {2008},
}