Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1185252
- author
- Zumarraga, Miren ; Camarero, Susana ; Shleev, Sergey LU ; Martinez-Arias, Arturo ; Ballesteros, Antonio ; Plou, Francisco J and Alcalde, Miguel
- organization
- publishing date
- 2008
- 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 Inc.
- external identifiers
-
- wos:000254035500024
- scopus:40549139786
- pmid:17932916
- ISSN
- 0887-3585
- DOI
- 10.1002/prot.21699
- language
- English
- LU publication?
- yes
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Analytical Chemistry (S/LTH) (011001004)
- id
- 7ccae9bd-1615-4b8b-8c1f-4cc969d9314f (old id 1185252)
- date added to LUP
- 2016-04-01 14:10:39
- date last changed
- 2022-01-27 23:12:18
@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}},
keywords = {{protein folding; C-terminal tail; in vivo assembly; laccase}},
language = {{eng}},
number = {{1}},
pages = {{250--260}},
publisher = {{John Wiley & Sons Inc.}},
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}},
doi = {{10.1002/prot.21699}},
volume = {{71}},
year = {{2008}},
}