Investigation On The Mechanism Of Dynamic Substrate Tunnel Modulation In Cytochrome P450s

The performance of the substrate access tunnel is one of the key factors affecting enzyme catalytic properties.Different from the previous substrate tunnel morphology analysis based on static structure,this work explored the dynamic effect of loop regions in cytochrome P450 on substrate access tunnel,and firstly proposed a loop engineering strategy to improve the transport properties and opening time of the cytochrome P450 substrate tunnel,revealing the molecular mechanism between the loop regions surrounding the substrate tunnel entrance and substrate tunnel.Firstly,molecular dynamics simulations and substrate access tunnel analysis have been carried out on CYP116B3 wild-type,variant 70A08,and variant 74H10.By comprehensive computational analysis,5 potentially functional positions（A86,T91,M108,A109,T111）on loop regions（loop B-B’and loop B’-C）have been identified and then subjected to simultaneously saturated mutagenesis.After screening and rescreening,9 beneficial variants were identified,among which the best variant 8G8（A86T/T91L/M108N/A109M/T111A）showed a 134-fold increase in catalytic efficiency(k_cat/K_m)and a coupling efficiency increase of about 5 times towards substrate 7-ethoxycoumarin compared to CYP116B3 wild-type.Additionally,variant 8G8 also exhibited significant improvement in the hydroxylation activity of polycyclic aromatic hydrocarbons,with a 9-fold increased hydroxylation activity for naphthalene,and more than 2.5-fold increased catalytic activity for toluene,acenaphthene,and fluorene.Subsequently,molecular dynamics simulations and dynamic tunnel analysis were performed on CYP116B3 wild-type and 9 beneficial variants.Computational analysis showed that reducing the flexibility of loop B-B’and loop B’-C around the tunnel entrance is beneficial for increasing the opening time of the substrate access tunnel and facilitating the substrate/product transport efficiency.In summary,this work proposed a powerful and universal loop engineering strategy for redesigning loop regions around the tunnel entrance to regulate the transport of substrate.