title: Activity improvement of a Kluyveromyces lactis aldo-keto reductase KlAKR via rational design.
abstract: Optically pure t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate ((R)-1b) is the key chiral precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. Wild-type aldo-keto reductase KlAKR from Kluyveromyces lactis has ideal diastereoselectivity toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a, dep>99.5%) but poor activity. A rational engineering was used to improve the KlAKR activity. Based on homology modeling and molecular docking, two amino acid residues (295 and 296) were selected as mutation sites, and two rounds of site-saturation mutagenesis were performed. Among the mutants, KlAKR-Y295W/W296L exhibited the highest catalytic efficiency (kcat/Km) toward 1a up to 12.37s(-1)mM(-1), which was 11.25-fold higher than that of wild-type KlAKR. Moreover, the majority of mutations have no negative impact on stereoselectivity. Using KlAKR-Y295W/W296L coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for cofactor regeneration, (R)-1b was accumulated up to 162.7mM with dep value above 99.5%. KlAKR-Y295W/W296L represents a robust tool for (R)-1b synthesis.
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China.
authors.forename: Xi
authors.initials: X
authors.lastname: Luo
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China.
authors.forename: Ya-Jun
authors.initials: YJ
authors.lastname: Wang
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China. Electronic address: zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D019943:Amino Acid Substitution; D002384:Catalysis; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase
keywords: Aldo-keto reductase; Atorvastatin calcium; Rational engineering; Site-saturation mutagenesis; T-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate