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 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.
Katzberg
Michael
M
Institute of Technical Chemistry and Biotechnology, Freiberg University of Mining and Technology, Germany.
Skorupa-Parachin
Nàdia
N
Gorwa-Grauslund
Marie-Françoise
MF
Bertau
Martin
M
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D005818:Genetic Engineering; D006588:Hexanones / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007659:Ketones / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
D006588:Hexanones; D007659:Ketones; D029701:Saccharomyces cerevisiae Proteins; D009249:NADP; C011269:2,5-hexanedione; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C432953:GRE2 protein, S cerevisiae