Rational design of Kluyveromyces marxianus ZJB14056 aldo –keto reductase KmAKR to enhance diastereoselectivity and activity

Publication date: Available online 1 August 2017 Source:Enzyme and Microbial Technology Author(s): Ya-Jun Wang, Bin-Bin Ying, Wei Shen, Ren-Chao Zheng, Yu-Guo Zheng t−Butyl 6−cyano−(3R,5R)−dihydroxyhexanoate ((3R,5R)−1b) is a valuable chiral synthon of atorvastatin calcium. A novel NADPH-specific aldo−keto reductase (AKR) was identified from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 by genome database mining, displaying t−butyl 6−cyano−(5R)−hydroxy−3−oxohexanoate ((5R)−1a) reducing activity and moderate diastereoselectivity (de p ∼80.5%). Molecular homology modeling and docking studies demonstrated that the side chain of Trp297 blocks binding of (5R)−1a to KmAKR. The mutation of Trp297 to His led to dramatic conformational changes and significant improvement in both diastereoselectivity and activity. In comparison with KmAKR, KmAKR−W297H displayed strict diastereoselectivity, and 2.8−fold, 3.9−fold improvement in k cat and k cat/K m toward (5R)−1a, which were 10.36s−1 and 6.56s−1·mM−1 respectively. Coupling KmAKR−W297H with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for coenzyme regeneration, 100mM (5R)−1a was completely reduced to (3R,5R)−1b within 12h, in a de p >99.5%. Graphical abstract
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research