Engineering of versatile redox partner fusions that support monooxygenase activity of functionally diverse cytochrome P450s

Abstract

Most bacterial cytochrome P450 monooxygenases (P450s or CYPs) require two redox partner proteins for activity. To reduce complexity of the redox chain, the Bacillus subtilis flavodoxin YkuN (Y) was fused to the Escherichia coli flavodoxin reductase Fpr (R), and activity was tuned by placing flexible (GGGGS)_n or rigid ([E/L]PPPP)_n linkers (n = 1-5) in between. P-linker constructs typically outperformed their G-linker counterparts, with superior performance of YR-P5, which carries linker ([E/L]PPPP)₅. Molecular dynamics simulations demonstrated that ([E/L]PPPP)_n linkers are intrinsically rigid, whereas (GGGGS)_n linkers are highly flexible and biochemical experiments suggest a higher degree of separation between the fusion partners in case of long rigid P-linkers. The catalytic properties of the individual redox partners were best preserved in the YR-P5 construct. In comparison to the separate redox partners, YR-P5 exhibited attenuated rates of NADPH oxidation and heme iron (III) reduction, while coupling efficiency was improved (28% vs. 49% coupling with B. subtilis CYP109B1, and 44% vs. 50% with Thermobifida fusca CYP154E1). In addition, YR-P5 supported monooxygenase activity of the CYP106A2 from Bacillus megaterium and bovine CYP21A2. The versatile YR-P5 may serve as a non-physiological electron transfer system for exploitation of the catalytic potential of other P450s.

Figure 1

Myristic acid conversion by B. subtilis CYP109B1 supported by different redox partners. Reactions were started by the addition of a mixture of NADPH and myristic acid and allowed to proceed for 120 min under the support of an NADPH regenerating system. (a) CYP109B1 (1 µM) conversion reactions were carried out in the presence of non-fused redox partners Fpr and FldA (black bars), Fpr and YkuN (white bars) or with different YkuN-Fpr fusion constructs (grey bars). Reductase (Fpr) and flavodoxin (FldA or YkuN) together with CYP109B1 were employed at respective ratios of 1:1:1, 1:10:1 and 4:4:1. Reactions conducted with YkuN-Fpr (YR) fusion constructs and CYP109B1 were carried out at a respective ratio of 4:1. YR indicates the linker-less YkuN-Fpr fusion construct, whereas linker designations P1 - P5 and G1 - G5 correspond to linker sequences (GGGGS)_n and ([E/L]PPPP)_n of different lengths (n = 1–5). The data presented are average values of 3–6 independent conversion reactions with indicated standard deviation. (b) Myristic acid conversion by CYP109B1 in the presence of different concentrations of selected fusion constructs or non-fused Fpr/YkuN. The ratio of non-fused redox partners was maintained at 1:1.

Figure 2

MD simulations of the (GGGGS)_n and ([E/L]PPPP)_n linkers used to functionally connect YkuN and Fpr. (a) Structural snapshots of (GGGGS)_n and ([E/L]PPPP)_n linkers of length 1 and 5. Depicted in each panel are the starting conformations (left) and the lowest energy conformation found during the 20 ns MD simulations (right). Development of the radius of gyration (Rg) during MD simulations for (GGGGS)_n and ([E/L]PPPP)_n linkers of lengths (n = 1–5) are shown in (b) and (c), respectively. The strong decrease in Rg for (GGGGS)_n linkers reflects the hydrophobic collapse, whereas the ([E/L]PPPP)_n linkers remain in an extended conformation.

Figure 3

Influence of auxiliary YkuN on CYP109B1 catalysis driven by the different YkuN-Fpr fusion constructs. Myristic acid conversion with CYP109B1 (1 µM) was carried out in the presence of the different Ykun-Fpr fusion constructs either in the absence (- YkuN) or presence of additional YkuN (4 or 9 µM). In all cases reactions were started by the addition of a mixture of NADPH and myristic acid and allowed to proceed for 120 min under the support of an NADPH regenerating system. The Fpr/YkuN/CYP109B1 system reconstituted at 1:10:1 and 1:1:1 ratio, respectively achieved 96% and 60% conversion of myristic acid in 120 min.

Figure 4

Conversion of β-ionone by T. fusca YX CYP154E1 supported by selected fusion constructs. Reactions were carried out in the presence of 1 µM CYP154E1 and 4 µM of indicated fusion constructs. Control reactions with non-fused Fpr/YkuN were conducted with 1 µM CYP154E1 together with either 1 µM Fpr + 10 µM YkuN [1:10], or 4 µM Fpr + 4 µM YkuN [4:4]. In all cases the initial concentration of the substrate β-ionone was 2 mM. Reactions were started by the addition of NADP⁺ under support of an NADPH regenerating system and stopped after 1 h.

Figure 5

Conversion of progesterone by bovine CYP21A2 supported by Fpr/YkuN and their fused derivative YR-P5. In all cases reactions were carried out in the presence of 0.5 µM CYP21A2, 200 µM progesterone and 5 µM of indicated redox partner(s). Corresponding ratios of redox partner(s) relative to CYP21A2 are indicated in brackets. Reactions were started by the addition of NADPH and supported by a NADPH regenerating system. Reactions were stopped after 30 min.