Switch in Cofactor Specificity of a Baeyer-Villiger Monooxygenase

被引：46

作者：

Beier, Andy ^{[1
]}

Bordewick, Sven ^{[1
]}

Genz, Maika ^{[1
]}

Schmidt, Sandy ^{[1
]}

van den Bergh, Tom ^{[2
]}

Peters, Christin ^{[1
]}

Joosten, Henk-Jan ^{[2
]}

Bornscheuer, Uwe T. ^{[1
]}

机构：

[1] Ernst Moritz Arndt Univ Greifswald, Inst Biochem, Dept Biotechnol & Enzyme Catalysis, Felix Hausdorff Str 4, D-17487 Greifswald, Germany

[2] Bioprodict, Nieuwe Marktstr 54E, NL-6511 AA Nijmegen, Netherlands

来源：

CHEMBIOCHEM | 2016年 / 17卷 / 24期

关键词：

Baeyer-Villiger monooxygenases; cofactor specificity; enzyme catalysis; protein engineering; rational design; KETOL-ACID REDUCTOISOMERASE; ENZYME CASCADE SYNTHESIS; COENZYME SPECIFICITY; CYCLOHEXANONE MONOOXYGENASE; FORMATE DEHYDROGENASE; REDUCTASE; NADPH; DETERMINANTS; STABILITY; REDESIGN;

D O I：

10.1002/cbic.201600484

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79%) compared to NADPH (58%) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.

引用

页码：2312 / 2315

页数：4

共 37 条

[1]

[Anonymous], PROTEIN ENG HDB

[2]

Baeyer A., 1899, BER DTSCH CHEM GES, V32, P3625, DOI DOI 10.1002/CBER.189903203151

[3] Discovery, application and protein engineering of Baeyer-Villiger monooxygenases for organic synthesis [J].