Designing New Baeyer−Villiger Monooxygenases Using Restricted CASTing

• Published in: Journal of organic chemistry Vol. 71; no. 22; pp. 8431 - 8437
• Main Authors: Clouthier, Christopher M, Kayser, Margaret M, Reetz, Manfred T
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 27.10.2006; Amer Chemical Soc
• Subjects: Alicyclic compounds; Alicyclic compounds, terpenoids, prostaglandins, steroids; Amino Acid Sequence; Chemistry; Chemistry, Organic; Combinatorial Chemistry Techniques; Cyclohexanones - chemistry; Directed Molecular Evolution - methods; Exact sciences and technology; Heterocyclic compounds; Heterocyclic compounds with o, s, se, te hetero atom and condensed derivatives; Mixed Function Oxygenases - chemical synthesis; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis; Organic chemistry; Oxygenases - chemical synthesis; Oxygenases - chemistry; Oxygenases - genetics; Physical Sciences; Preparations and properties; Science & Technology; Stereoisomerism; Substrate Specificity - genetics; Time Factors; OXIDATION; SUBSTRATE ACCEPTANCE; GENE-CLUSTER; ENANTIOSELECTIVE BIOCATALYSTS; ENZYMES; FORCE-FIELD; CYCLOHEXANONE MONOOXYGENASE; SIMULTANEOUS IDENTIFICATION; DIRECTED EVOLUTION; RNA-POLYMERASE; Cyclopentanone monooxygenase; Catalytic reaction; Enantioselectivity; Enzyme; Active site; Lactone; Oxygen heterocycle; Oxygenase; Baeyer Villiger reaction; Enantiomeric excess; Oxidation; Oxidoreductases; Mutation
• ISSN: 0022-3263; 1520-6904
• DOI: 10.1021/jo0613636

This paper outlines the design and execution of the first mini-evolution of cyclopentanone monooxygenase (CPMO). The methodology described is a relatively inexpensive and rapid way to obtain mutant enzymes with the desired characteristics. Several successful mutants with enhanced enantioselectivities were identified. For example, mutant-catalyzed oxidation of 4-methoxycyclohexanone gave the corresponding lactone with 92% entantiometric excess (ee) compared to the 46% ee achieved with wild-type cyclohexanone monoxygenase (WT-CHMO). The original design of the mini-evolution and the following evaluation of mutants can provide valuable insights into the active site's construction and dynamics and can suggest other catalytically profitable mutations within the putative active site.