title: Anchoring effects in a wide binding pocket: the molecular basis of regioselectivity in engineered cytochrome P450 monooxygenase from B. megaterium.
abstract: The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megaterium (CYP102A1) with its flexible and widely opened active site is still not well understood. In the present work (-)-alpha-pinene bound complexes with two triple mutants were modeled to elucidate the contribution of the three major factors that mediate selectivity: active site shape, protein flexibility, and chemical reactivity of the substrate. For the triple mutant A74G F87V L188Q (GVQ), one stable, productive conformation of the substrate (conformation I) was identified by multiple molecular dynamics simulations. The model predicts pinene epoxide as a major product (42% pinene oxide, 23% verbenol) which is in agreement with the experimental product profile (70% pinene oxide, 20% verbenol). In contrast, for the triple mutant A74G F87G L188Q (GGQ) two stable productive substrate conformations were identified (conformations IIa and IIb), and verbenol was predicted as major product (81% verbenol, 16% myrtenol), which is in agreement with experimental results (77% verbenol, 10% myrtenol). The effect of chemical reactivity of the substrate was demonstrated by comparison of (-)-alpha-pinene to its regioisomer (-)-beta-pinene, where the product profile is shifted from 68% pinocarveol and 32% myrtanal in mutant GVQ, to 40% pinocarveol and 60% myrtanal in mutant GGQ. Our results strongly suggest a major role of residue 87 in anchoring (-)-alpha-pinene during substrate binding which provides a simple and elegant rationalization of the dynamic structure of this enzyme-substrate complex.
authors.affiliation: REQUIMTE, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, P 4169-007 Porto, Portugal.
authors.forename: Ricardo J F
authors.initials: RJ
authors.lastname: Branco
authors.forename: Alexander
authors.initials: A
authors.lastname: Seifert
authors.forename: Michael
authors.initials: M
authors.lastname: Budde
authors.forename: Vlada B
authors.initials: VB
authors.lastname: Urlacher
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Ramos
authors.forename: Jürgen
authors.initials: J
authors.lastname: Pleiss
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology*; D000081008:Bicyclic Monoterpenes; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D020134:Catalytic Domain; D003198:Computer Simulation; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008968:Molecular Conformation; D039821:Monoterpenes / Q000737:chemistry; D050505:Mutant Proteins / Q000737:chemistry; D010084:Oxidation-Reduction; D015202:Protein Engineering; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D000081008:Bicyclic Monoterpenes; D001643:Bridged Bicyclo Compounds; D039821:Monoterpenes; D050505:Mutant Proteins; C010789:beta-pinene; D003577:Cytochrome P-450 Enzyme System; C005451:alpha-pinene