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Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

Katzberg, Michael; Skorupa Parachin, Nadia LU ; Gorwa-Grauslund, Marie-Francoise LU and Bertau, Martin (2010) In International Journal of Molecular Sciences 11(4). p.1735-1758
Abstract
The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the gamma-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the gamma-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio-as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be... (More)
The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the gamma-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the gamma-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio-as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Gre2p, site-directed-mutagenesis, S. cerevisiae, biocatalysis, 5-hexanedione, 2, cofactor preference
in
International Journal of Molecular Sciences
volume
11
issue
4
pages
1735 - 1758
publisher
MOLECULAR DIVERSITY PRESERVATION INT
external identifiers
  • wos:000277119800024
  • scopus:77951894908
ISSN
1422-0067
DOI
10.3390/ijms11041735
language
English
LU publication?
yes
id
a2dbde5a-1f58-4d30-8674-d9ec1095fad2 (old id 1619426)
date added to LUP
2010-06-18 12:42:41
date last changed
2018-05-29 09:45:28
@article{a2dbde5a-1f58-4d30-8674-d9ec1095fad2,
  abstract     = {The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the gamma-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the gamma-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio-as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.},
  author       = {Katzberg, Michael and Skorupa Parachin, Nadia and Gorwa-Grauslund, Marie-Francoise and Bertau, Martin},
  issn         = {1422-0067},
  keyword      = {Gre2p,site-directed-mutagenesis,S. cerevisiae,biocatalysis,5-hexanedione,2,cofactor preference},
  language     = {eng},
  number       = {4},
  pages        = {1735--1758},
  publisher    = {MOLECULAR DIVERSITY PRESERVATION INT},
  series       = {International Journal of Molecular Sciences},
  title        = {Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example},
  url          = {http://dx.doi.org/10.3390/ijms11041735},
  volume       = {11},
  year         = {2010},
}