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Biochim Biophys Acta. 2016 Dec;1864(12):1641-1648. doi: 10.1016/j.bbapap.2016.08.015. Epub 2016 Aug 26.

The role of conformational flexibility in Baeyer-Villiger monooxygenase catalysis and structure.

Author information

1
Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Bellini Pavilion, Room 466, Montreal, Quebec H3G 0B1, Canada; Groupes de recherche GRASP et PROTEO, Montreal, Quebec, Canada.
2
Departments of Microbiology & Immunology and Chemistry, McGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada; National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Zone, Tianjin 300308, China; FQRNT Center for Green Chemistry and Catalysis, Montreal, Quebec, Canada.
3
Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Bellini Pavilion, Room 466, Montreal, Quebec H3G 0B1, Canada; Department of Microbiology & Immunology, McGill University, 3649 Promenade Sir William Osler, Bellini Pavilion, Room 466, Montreal, Quebec H3G 0B1, Canada; Groupes de recherche GRASP et PROTEO, Montreal, Quebec, Canada. Electronic address: albert.berghuis@mcgill.ca.

Abstract

BACKGROUND:

The Baeyer-Villiger monooxygenases (BMVOs) are a group of microbial enzymes that have garnered interest as industrial biocatalysts. While great strides have been made in recent years to understand the mechanism of these enzymes from a structural perspective, our understanding remains incomplete. In particular, the role of a twenty residue loop (residues 487-504), which we refer to as the "Control Loop," that is observed in either an ordered or disordered state in various crystal structures remains unclear.

METHODS:

Using SAXS, we have made the first observations of the Loop in solution with two BVMOs, cyclohexanone monooxygenase (CHMO) and cyclopentadecanone monooxygenase. We also made a series of mutants of CHMO and analyzed them using SAXS, ITC, and an uncoupling assay.

RESULTS:

These experiments show that Control Loop ordering results in an overall more compact enzyme without altering global protein foldedness. We have also demonstrated that the Loop plays a critical and complex role on enzyme structure and catalysis. The Control Loop appears to have a direct impact on the organization of the overall structure of the protein, as well as in influencing the active site environment.

CONCLUSIONS:

The data imply that the Loop can be divided into two regions, referred to as "sub-loops," that coordinate overall domain movements to changes in the active site.

GENERAL SIGNIFICANCE:

A better understanding of the mechanistic role of the Control Loop may ultimately be helpful in designing mutants with altered specificity and improved catalytic efficiency.

KEYWORDS:

Baeyer-Villiger monooxygenase; Enzyme catalysis; Enzyme mechanism; Isothermal titration calorimetry (ITC); Protein flexibility; Small-angle X-ray scattering (SAXS)

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