Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design.
Bioorg Chem
; 90: 103018, 2019 09.
Article
em En
| MEDLINE
| ID: mdl-31220674
t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The Km values of M3 for NADPH and (5R)-1 were 0.15â¯mmol/L and 1.41â¯mmol/L, and the maximal reaction rate vmax was 55.56⯵mol/min/mg. Compared with M1, the catalytic efficiency kcat/Km of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5â¯h to completely reduce (5R)-1 at up to 100.0â¯g/L, producing 237.4â¯mmol/L (3R,5R)-2 in d.e.P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8â¯g/L/d.
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01-internacional
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MEDLINE
Assunto principal:
Caproatos
/
Proteínas Fúngicas
/
Aldo-Ceto Redutases
Idioma:
En
Revista:
Bioorg Chem
Ano de publicação:
2019
Tipo de documento:
Article