Three pairs of surrogate redox partners comparison for Class I cytochrome P450 enzyme activity reconstitution

Abstract

Most P450s require redox partners for the electron transfer during catalysis. However, little information is available on cognate redox partners for P450s, which greatly limits P450 function exploration and practical application. Thus, the stategy of building various hybrid P450 catalytic systems with surrogate redox partner has often adopted to engineer P450 biocatalysts. In this study, we compare three pairs of frequently-used surrogate redox partner SelFdx1499/SelFdR0978, Adx/AdR and Pdx/PdR and in terms of their electron transfer properties. The three selected bacterial Class I P450s include PikC, P450sca-2 and CYP-sb21, which are responsible for production of high-value-added products. Here we show that SelFdx1499/SelFdR0978 is the most promising redox partner compared to Adx/AdR and Pdx/PdR. The results provide insights into the domination for P450-redox partner interactions in modulating the catalytic activity of P450s. This study not only produces a more active biocatalyst but also suggests a general chose for a universal reductase which would facilitate engineering of P450 catalyst.

Cooperative Substrate Binding Controls Catalysis in Bacterial Cytochrome P450terp (CYP108A1)

“…As more P450s were discovered and the number of detailed mechanistic studies expanded, it became clear that P450cam exhibits some additional unusual regulatory properties. The earliest unique feature of P450cam to be discovered was its strict requirement for its own redox partner, while many other P450s can be supported by foreign redox partners. − We now know that Pdx binding to P450cam results in a Pdx-specific induced conformational change that triggers the formation of a proton relay network required for O 2 activation. − More recently, P450cam has been found to have a second allosteric substrate binding site and undergoes complex and large structural changes in response to the binding of heme iron ligands. , …”

Efficient hydroxylation of flavonoids by using whole-cell P450 sca-2 biocatalyst in Escherichia coli

“…Notably, the E. coli flavodoxin Fld was used in combination with Fpr for the first time to reconstitute the activity of P450s. In the recent study, FdR_0978/Fdx_1499 was the most promising redox partner for the in vitro activity of sca-2 towards mevastatin compared to the redox systems Adx/AdR and Pdx/PdR ( Liu et al, 2022 ). However, the HFLA-6 strain (harboring plasmid pRSF-sca-2 mut -Fdx_1499-FdR_0978) produced 8.9 ± 0.2 and 9.7 ± 0.6 mg/L eriodictyol in the in vivo biocatalytic system containing glycerol or glucose, which were only 23.1% and 31.2% of the HFLA-7 strain, respectively ( Figure 3B ).…”

Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts

“…Our data shows that ferredoxin provides optimal coupling of CYP2A_B49 to photosynthetic reducing power. Although coupling P450 activity to photosynthetic reducing power is still a relatively niche field, plant and cyanobacterial ferredoxins has been used as surrogate electron transfer partners in many reconstituted P450 systems ( Wirtz et al., 2000 ; Jackson et al., 2002 ; Goñi et al., 2009 ; Jensen et al., 2012 ; Zhang et al., 2018 ; Mie et al., 2020 ; Zhu et al., 2020 ; Liu et al., 2022 ). Recently a survey of 16 microbial ferredoxins found ferredoxin from the cyanobacteria Synechococcus elongatus PCC 7942 to be the most efficient redox partner for 5 different P450s ( Zhang et al., 2018 ).…”

“…Recently a survey of 16 microbial ferredoxins found ferredoxin from the cyanobacteria Synechococcus elongatus PCC 7942 to be the most efficient redox partner for 5 different P450s ( Zhang et al., 2018 ). This was later expanded to include further 2 bacterial P450s favoring non-native cyanobacterial ferredoxin redox partners ( Liu et al., 2022 ). Together with our own findings, these results suggest that plant and cyanobacterial [2Fe-2S] ferredoxins may be ideal generalist P450 redox partners.…”