Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example
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 Sa...
Main Authors: | , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
MDPI AG
2010-04-01
|
Series: | International Journal of Molecular Sciences |
Subjects: | |
Online Access: | http://www.mdpi.com/1422-0067/11/4/1735/ |
id |
doaj-0113e8f250ae47d9a5ef7b819b728ba7 |
---|---|
record_format |
Article |
spelling |
doaj-0113e8f250ae47d9a5ef7b819b728ba72020-11-24T22:02:43ZengMDPI AGInternational Journal of Molecular Sciences1422-00672010-04-011141735175810.3390/ijms11041735Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an ExampleMichael KatzbergNàdia Skorupa-ParachinMarie-Françoise Gorwa-GrauslundMartin BertauThe 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 γ-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the γ-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. http://www.mdpi.com/1422-0067/11/4/1735/biocatalysis2,5-hexanedioneS. cerevisiaesite-directed-mutagenesisGre2pcofactor preference |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Michael Katzberg Nàdia Skorupa-Parachin Marie-Françoise Gorwa-Grauslund Martin Bertau |
spellingShingle |
Michael Katzberg Nàdia Skorupa-Parachin Marie-Françoise Gorwa-Grauslund Martin Bertau Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example International Journal of Molecular Sciences biocatalysis 2,5-hexanedione S. cerevisiae site-directed-mutagenesis Gre2p cofactor preference |
author_facet |
Michael Katzberg Nàdia Skorupa-Parachin Marie-Françoise Gorwa-Grauslund Martin Bertau |
author_sort |
Michael Katzberg |
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 |
title_short |
Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example |
title_full |
Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example |
title_fullStr |
Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example |
title_full_unstemmed |
Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example |
title_sort |
engineering cofactor preference of ketone reducing biocatalysts: a mutagenesis study on a γ-diketone reductase from the yeast saccharomyces cerevisiae serving as an example |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2010-04-01 |
description |
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 γ-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the γ-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. |
topic |
biocatalysis 2,5-hexanedione S. cerevisiae site-directed-mutagenesis Gre2p cofactor preference |
url |
http://www.mdpi.com/1422-0067/11/4/1735/ |
work_keys_str_mv |
AT michaelkatzberg engineeringcofactorpreferenceofketonereducingbiocatalystsamutagenesisstudyonagdiketonereductasefromtheyeastsaccharomycescerevisiaeservingasanexample AT nadiaskorupaparachin engineeringcofactorpreferenceofketonereducingbiocatalystsamutagenesisstudyonagdiketonereductasefromtheyeastsaccharomycescerevisiaeservingasanexample AT mariefrancoisegorwagrauslund engineeringcofactorpreferenceofketonereducingbiocatalystsamutagenesisstudyonagdiketonereductasefromtheyeastsaccharomycescerevisiaeservingasanexample AT martinbertau engineeringcofactorpreferenceofketonereducingbiocatalystsamutagenesisstudyonagdiketonereductasefromtheyeastsaccharomycescerevisiaeservingasanexample |
_version_ |
1725834375758610432 |