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.