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.

Fig. 1. The P450-catalyzed reactions.

a PikC hydroxylates YC-17 to produce the mono-hydroxylated products methymycin and neomethymycin, and two dioxygenation products novamethymycin and ketomethymycin. b P450sca-2 hydroxylates mevastatin to form pravastatin and 6-epi-pravastatin. c CYP-sb21 catalyzes the oxidation of cyclosporine A (CsA) to γ-hydroxy-N-methyl-L-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-L-Leu9-CsA (CsA-9-OH). Functional groups and bonds colored in red are catalyzed by the P450s.

Fig. 2. Influence of the selection of redox partner on P450 activities.

The catalytic activities of PikC (a), P450sca-2 (b), and CYP-sb21 (c) when supported by nine different combinations of redoxin and redoxin reductase. All the experiments were carried out in triplicate. The error line represents the standard deviation. P values in each groups were calculated with single factor ANOVA analysis, and all P values were <0.01.

Fig. 3. DCIP and cyt c reduction assays.

a DCIP reduction activities of the three redoxin reductases in the presence of 1 mM NADH or NADPH. b Rates of electron transfer measured by cyt c reduction. The reaction mixture contained 30 μM cyt c, 10 nM FdR or 10 nM FdR coupled with 20 nM Fdx, and started by adding 500 μM NAD(P)H. All experiments were independently repeated for three times. The error line represents the standard deviation. P values in each groups were calculated with single factor ANOVA analysis, and all P values were <0.01.

Fig. 4. Electrostatic surface analysis of P450s and Fdxs.

Positively and negatively charged surfaces are colored in blue and red, respectively. The electrostatic surfaces of Fdxs with Helix1 (H1), Helix2 (H2), or Helix3 (H3) are marked. The six proteins are PikC/YC-17 (PDB ID: 2VZ7), P450sca-2 (predicted by Alphafold2), CYP-sb21 (substrate-free, PDB ID: 6M4S), SelFdx1499 (predicted by Alphafold2), Adx (PDB ID: 1CJZ), and Pdx (PDB ID: 1PDX).

Fig. 5. Fdx-P450 docking models.

Docking models of three Fdxs in complex with PikC (a), P450sca-2 (b), and CYP-sb21 (c). The structures of P450-Fdx complexes are shown as cartoon with different colors. The key interacting residues on P450-Fdx interfaces are shown as sticks in yellow and green, respectively. Heme and substrates are shown as sticks in red and wheat, respectively. The Fe₂S₂ cluster are shown as spheres. The distances (Å) between the iron–sulfur cluster and heme-iron are indicated by dashed red lines.

Fig. 6. Fdx-FdR docking models.

Docking models of three FdRs in complex with SelFdx1499 (a), Adx (b), and Pdx (c). The structures of FdR-Fdx complexes are shown as cartoon with different colors. The key interacting residues on FdR-Fdx interfaces are shown as sticks in yellow and green, respectively. FAD is shown as sticks in cyan. The Fe₂S₂ cluster is shown as spheres. The shortest FAD-Fe₂S₂ distances (Å) are indicated by dashed red lines.