Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

Abstract

This paper reports the structure of the double mutant Asp251Gly/Gln307His (here called A2), generated by random mutagenesis, is able to produce 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. Here, we report the crystal structure of the heme domain of the mutant in the substrate-free form and in complex with the substrate N-palmitoylglycine, together with its impact on thermal stability, reduction potential and electron transfer.The substrate-free structure adopts a conformation more similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop, part of the substrate access channel. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on Ihelix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules.Differential scanning calorimetry shows a population destabilized by 2.2°C compared to the wild type protein, probably reflecting the increased flexibility of part of the protein compared to WT. Moreover, a shift of the heme reduction potential by 50 mV toward positive values, a 2-folds higher first electron transfer rate in the absence of oxygen and a 4-folds higher NADPH consumption rate in the presence of oxygen as the only electron acceptors, when compared to wild type protein.The data demonstrate that a single mutation far away from the active site is able to trigger an increase in protein flexibility in a key region of the substrate access channel, shifting the conformational equilibrium toward the closed form of the protein that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted.

Working at the membrane interface: Ligand‐induced changes in dynamic conformation and oligomeric structure in human aromatase

“…C). It is known that these elements, particularly the F‐G loop, are flexible in cytochromes P450 and allow substrate entrance along the access channel . The RMSF profile obtained from our 20 nSec simulation is in very good agreement with the one previously reported for human aromatase (250 nSec) .…”

Progress in biopharmaceutical development

“…By contrast, P450 BM3, bacterial P450 enzyme from Bacillus megaterium , has been shown to be able to produce drug metabolites typical of the human enzymes with a high coupling efficiency . Owing to P450 BM3 characteristics, multiple protein engineering studies have been performed on this enzyme to widen its catalytic abilities . Moreover, several constructions have been already reported including different fusion human P450 enzymes, engineered by connecting the P450 BM3 reductase domain with human cytochromes P450 3A4, 2C9, 2C19 , 2A6, CYP2C6, and CYP4F11 , monkey 2C20 , and dog CYP2D15 .…”

Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras

“…This has led to a wide range of protein engineering studies on this enzyme to widen its catalytic abilities (Tsotsou et al, 2012, 2013; Ryu et al, 2014; Di Nardo et al, 2015; Ren et al, 2015; Capoferri et al, 2016). …”