Anchoring effects in a wide binding pocket: The molecular basis of regioselectivity in engineered cytochrome P450 monooxygenase from B. megaterium

• Tipo: Artigo em Revista Científica Internacional
• Data: 2008

Autores

Ricardo J F Branco (Autor) Outra; Alexander Seifert (Autor) Outra; Michael Budde (Autor) Outra; Vlada B Urlacher (Autor) Outra; Maria J Ramos (Autor) FCUP; Juergen Pleiss (Autor) Outra

Revista

Título: Proteins: Structure, Function and Genetics

Vol. 73

Páginas: 597-607

ISSN: 0887-3585

Editora: Wiley-Blackwell

Indexação

ISI Web of Knowledge - 23 Citações

Scopus - 28 Citações

Classificação Científica

FOS: Ciências exactas e naturais > Ciências biológicas

Outras Informações

ID Authenticus: P-003-TX8

DOI: 10.1002/prot.22083

Abstract (EN): The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megaterium (CYPI02A1) 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 provdes a simple and elegant rationalization of the dynamic structure of this enzyme-substrate complex.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 11