title: Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue.
abstract: 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.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Hugo L
authors.initials: HL
authors.lastname: van Beek
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Hein J
authors.initials: HJ
authors.lastname: Wijma
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Lucie
authors.initials: L
authors.lastname: Fromont
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
keywords: BVMO, Baeyer–Villiger monooxygenase; Baeyer–Villiger monooxygenase; CHMO, cyclohexanone monooxygenase; Computational design; DTT, dithiothreitol; Disulfide bonds; Enzyme engineering; Flavoprotein; MD, molecular dynamics; PAMO, phenylacetone monooxygenase; Thermostability