Design and improvement of artificial redox modules by molecular fusion of flavodoxin and flavodoxin reductase from Escherichia coli

Abstract

A variety of fusion proteins between the versatile redox partners flavodoxin (FldA) and flavodoxin reductase (Fpr) from Escherichia coli was constructed with the aim to improve the electron transfer properties. The order in which FldA and Fpr were fused and the linker region between them was varied in a systematic manner. A simple molecular tool, designated “DuaLinX”, was developed that facilitated the parallel introduction of flexible glycine-rich and rigid proline-rich linkers between the fusion partners in a single cloning event. The fusion constructs were tested for their ability to transfer electrons to cytochrome c and cytochrome P450 109B1 from Bacillus subtilis. With CYP109B1, the performance of the constructs showed, independent of the domain order, a strong dependency on linker length, whereas with cytochrome c this phenomenon was less pronounced. Constructs carrying linkers of ≥15 residues effectively supported the CYP109B1-catalysed hydroxylation of myristic acid. Constructs carrying proline-rich linkers generally outperformed their glycine-rich counterparts. The best construct, FldA-Fpr carrying linker ([E/L]PPPP)4, supported CYP109B1 activity equally well as equivalent amounts of the non-fused redox partners, while cytochrome c reductase activity was ~2.7-fold improved. Thus, to functionally connect redox partners, rigid proline-rich linkers may be attractive alternatives to the commonly used flexible glycine-rich linkers.

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

“…Insertion of a suitable linker between the fusion partners however, substantially improved the overall performance. Herein, long ([E/L]PPPP) n linkers (n = 4–5) were particularly effective (Figs 1 and 4 ), which is consistent with previous findings for fusions between E. coli FldA and Fpr 19 . At protein concentrations ≥4 µM, the superior construct YR-P5 acted as a nearly equivalent substitute for non-fused Fpr/YkuN in driving CYP109B1 catalysis (Fig.…”

“…YkuN was fused to the N-terminus of Fpr, leaving the C-terminus of Fpr free, as it was demonstrated previously that attachment of FldA to the C-terminus of Fpr leads to reduced enzyme function, whereas constructs with the fusion partners in reversed order were substantially more active 19 . Attachment of the flavodoxin to the C-terminus of Fpr likely interferes with the function of the aromatic residues at the extreme C-terminus of Fpr, which are important for NADPH binding and electron exchange 30 – 32 .…”

“…To simplify redox chains and to improve the catalytic properties of P450 systems, a variety of man-made P450 fusion enzymes have been successfully created using a variety of molecular approaches, including “Molecular Lego” 22 , “LICRED” 56 , and”PUPPET” 57 . Here, the versatility and superior properties of YkuN were exploited to generate redox fusion enzymes capable of driving catalysis of different CYPs, using the previously established DuaLinX procedure 19 for linker engineering. The catalytic performance of a reconstituted P450 system is often limited by a low coupling efficiency.…”

“…To tune activity of the fused redox partners the linker region between them was varied in a systematic manner. Herein, both flexible (GGGGS) n and rigid ([E/L]PPPP) n linkers of different lengths (n = 1–5) were introduced between YkuN and Fpr by using the previously established DuaLinX tool 19 . After expression in E. coli and subsequent purification, the activity of the fusion constructs was investigated with respect to their ability to oxidize NADPH and reduce the P450 heme iron, as well as their capability to support the monooxygenase activity of a variety of P450s in vitro .…”

Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450

“…However, when we applied tethering strategies to increase the affinity of the Cytochrome P450 and the redox partners towards each other, we observed higher titers with the non-natural redox partners. Tethering strategies have previously been applied to several Cytochrome P450 enzymes, both in vitro [35,42,[48][49][50][51] and in vivo [42,48]. The in vitro studies showed that tethered redox complexes are able to overcome the need to use an excess of redox partners over the Cytochrome P450 enzyme, to compensate for low protein-protein affinities (typically a five-to 20-fold molar excess of ferredoxin is used in vitro).…”