Designing new Baeyer-Villiger monooxygenases using restricted CASTing.
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.
Clouthier
Christopher M
CM
Department of Physical Sciences, University of New Brunswick, Saint John, New Brunswick, E2L 4L5 Canada.
Kayser
Margaret M
MM
Reetz
Manfred T
MT
D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques; D003512:Cyclohexanones / Q000737:chemistry*; D019020:Directed Molecular Evolution / Q000379:methods*; D006899:Mixed Function Oxygenases / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D010105:Oxygenases / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000235:genetics; D013997:Time Factors
C521689:4-methoxycyclohexanone; D003512:Cyclohexanones; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C067215:cyclopentanone monooxygenase