title: The role of conformational flexibility in Baeyer-Villiger monooxygenase catalysis and structure.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors: 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.
authors: Brahm J
authors: BJ
authors: Yachnin
authors: 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.
authors: Peter C K
authors: PCK
authors: Lau
authors: 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.
authors: Albert M
authors: AM
authors: Berghuis
mesh_terms: D000150:Acinetobacter / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002151:Calorimetry; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D009249:NADP; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C019961:cyclohexanone oxygenase
keywords: Baeyer-Villiger monooxygenase; Enzyme catalysis; Enzyme mechanism; Isothermal titration calorimetry (ITC); Protein flexibility; Small-angle X-ray scattering (SAXS)