Open AccessStabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue
Author(s) -
van Beek Hugo L.,
Wijma Hein J.,
Fromont Lucie,
Janssen Dick B.,
Fraaije Marco W.
Publication year - 2014
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1016/j.fob.2014.01.009
Subject(s) - thermostability , chemistry , cysteine , protein engineering , monooxygenase , residue (chemistry) , hydrogen bond , disulfide bond , enzyme , protein disulfide isomerase , stereochemistry , combinatorial chemistry , organic chemistry , biochemistry , molecule , cytochrome p450
Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer–Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C–A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.