Rational and semi-rational engineering of cytochrome P450s for biotechnological applications

Abstract

The cytochrome P450 enzymes are ubiquitous heme-thiolate proteins performing regioselective and stereoselective oxygenation reactions in cellular metabolism. Due to their broad substrate scope and catalytic versatility, P450 enzymes are also attractive candidates for many industrial and biopharmaceutical applications. For particular uses, enzyme properties of P450s can be further optimized through directed evolution, rational, and semi-rational engineering approaches, all of which introduce mutations within the P450 structures. In this review, we describe the recent applications of these P450 engineering approaches and highlight the key regions and residues that have been identified using such approaches. These “hotspots” lie within critical functional areas of the P450 structure, including the active site, the substrate access channel, and the redox partner interaction interface.

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

“…Maintenance of the P450 catalytic cycle relies on continuous electron transport to the heme-iron by redox partners, which are complicated electron-transfer systems. Based on the types of redox partners and the P450-redox partner interaction relationships, P450 systems can be divided into five main classes (10,11,15) (Fig. 2).…”

“…Protein engineering involves modification of the residues based on the folding principles and molecular structure of proteins, with the goal of obtaining the desired mutated proteins with enhanced properties to compensate for the poor stability, low selectivity, slow catalytic rates, and limited application space of the native proteins (51). Directed evolution and rational and semi-rational design are routinely used methods in JBC REVIEWS: Engineering of P450 systems P450 engineering and play very important roles in the development of pharmaceutical catalysts (15,52).…”

“…Rational or semi-rational design, based on the well-characterized protein tertiary structure and the mechanistic understanding of structure-activity relationships, is regarded as an effective alternate strategy. Generally (but not always), "hot spot" residues for rational and semi-rational design are usually located within the SRS, the substrate access channel, and the P450-redox partner interaction sites (15,61).…”

Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana

“…Protein rational design can effectively reduce the number of variants to be generated, which in turn significantly reduce the screening workload. However, rational design is usually limited to the enzymes whose structure-activity relationships are well-established [ 15 , 49 ]. Because we could not solve the co-crystallization conditions, AutoDock and MD analyses were therefore used to explore potential CsA binding poses.…”

Cytochrome P450 Metabolism of Polyunsaturated Fatty Acids and Neurodegeneration

“…The final product depends on the specific CYP enzyme, as well as the type of PUFA substrate. In general, the regio/stereoselectivity, catalytic properties, and structure of each CYP enzyme might be significantly determined by a B-C loop in their structure, controlling the type of reaction and prevalence of a specific regio/stereoisomer product [ 56 , 74 , 75 ]. In this section, we discuss the major CYPs and their products when ω-3 and ω-6 PUFAs are substrates.…”