RESUMO
Catching the structure of cytochromeâ P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.
Assuntos
Sistema Enzimático do Citocromo P-450 , Estirenos , Oxirredução , Sistema Enzimático do Citocromo P-450/metabolismo , CatáliseRESUMO
Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure-based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N-abietoyl-l-tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild-type P450BM3 haem domain, with visible crystals forming within 2â hours and diffracting to a near-atomic resolution of 1.22â Å. Using these crystals as seeds in a cross-microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem-domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.
Assuntos
Proteínas de Bactérias/química , Cristalização/métodos , Sistema Enzimático do Citocromo P-450/química , NADPH-Ferri-Hemoproteína Redutase/química , Bacillus megaterium/química , Catálise , Heme/química , Indicadores e Reagentes , Metaloporfirinas/síntese química , Mutagênese Sítio-Dirigida , Ligação Proteica , Especificidade por Substrato , Difração de Raios XRESUMO
Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.