Directed evolution of P450cin for mediated electron transfer

Abstract

Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C-H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions. However, natural enzymes are often not suitable for using non-natural electron delivery systems. Here we report the directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III)sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of Sequence Saturation Mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generated a variant (Gln385His, Val386Ser, Thr77Asn, Leu88Arg; named KB8) with a 3.8-fold increase in catalytic efficiency (28 µM min) compared to P450cin-ADD-CinC (7 µM min). Furthermore, variant KB8 exhibited a 1.5-fold higher product formation (500 µM µM P450) compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as co-factor (315 µM µM P450). In addition, electrochemical experiments with the electron delivery system platinum/cobalt(III)sepulchrate showed that the KB8 variant had a 4-fold higher product formation rate (0.16 nmol (nmol) P450 min cm) than the P450cin-ADD-CinC (0.04 nmol (nmol) P450 min cm). In summary, the current work shows prospects of using directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems.

Review: Engineering of thermostable enzymes for industrial applications

“…In fact, directed evolution generates, and selects under a specific evolutionary pressure, enzymes with novel or improved features through an iterative process characterized by several rounds of mutagenesis and screening, starting from a parental protein. Generally, the creation of random mutants from a parental protein is done by error prone mutagenesis, 55–57 DNA shuffling, 58–60 site-saturation mutagenesis, 61,62 chemical mutagenesis, 63,64 or using different mutator strains 65–67 . However, all these methods generally require reiterative manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes.…”

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

“…Electrochemical reductions have been used to circumvent the requirement for redox partners in shuttling electrons from NADPH to P450, with the electrode being the source of reducing equivalents. Progress with electrode-adsorbed/immobilization of P450 enzymes on various electrodes has been accomplished by engineering of both electrodes and enzymes, including layer-by-layer films with polyions (129,130), a cobalt(III) sepulchrate (Zn/CoIIIsep) mediator (131,132), covalent immobilization to a gold (Au) self-assembled monolayer (133), and nanomaterial-modified electrodes (134,135). Due to the limitations of applying purified soluble P450s on various electrodes, protein film electrochemistry has been considered in electrocatalytic studies.…”

Nonconventional regeneration of redox enzymes – a practical approach for organic synthesis?

“…70 Also increasing the electron transfer rate between the mediator molecule and the P450 heme-site represents a very promising approach to address the Oxygen Dilemma. [84][85][86][87][88][89][90][91][92] Mechanistically, photochemical reduction is very similar to electrochemical regeneration of P450 monooxygenases. Hence it is not very astonishing that also (in)direct photochemical regeneration of P450 monooxygenases has been investigated.…”