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Cytochrome P450 enzymes (CYPs) have attracted much promise as biocatalysts in a push for cleaner and more environmentally friendly catalytic systems. However, changing the substrate specificity of CYPs, such as CYP102A1, can be a challenging task, requiring laborious mutagenesis. An alternative approach is the use of decoy molecules that "trick" the enzyme into becoming active by impersonating the native substrate. Whilst the decoy molecule system has been extensively developed for CYP102A1, its general applicability for other CYP102-family enzymes has yet to be shown. Herein, we demonstrate that decoy molecules can "trick" CYP102A5 and A7 into becoming active and hydroxylating non-native substrates. Furthermore, significant differences in decoy molecule selectivity as well as decoy molecule binding were observed. The X-ray crystal structure of the CYP102A5 haem domain was solved at 2.8 Å, delivering insight into a potential substate-binding site that differs significantly from CYP102A1. Decoy molecules were shown to be compatible for use with both CYP102A5 and CYP102A7. Both CYP102A5 and CYP102A7 displayed a unique and unexpected decoy molecule preference. The crystal structure of CYP102A5 was solved at a resolution of 2.8 Å. [Display omitted] • Decoy molecules were shown to be compatible for use with both CYP102A5 and A7. • Both CYP102A5 and A7 displayed a unique and unexpected decoy molecule preference. • Decoy molecules were found to bind in a two-step fashion to CYP102A5 and A7 that was not observed for A1. • We report the first structure of CYP102A5 at a resolution of 2.8 Å. • The structure of CYP102A5 reveals a potential binding site that differs significantly from the CYP102A1 reference system. [ABSTRACT FROM AUTHOR]

To bring out the potential of wild-type cytochrome P450s, we have developed a series of 'decoy molecules' to change their high substrate specificity without any mutagenesis. Decoy molecules are inert dummy substrates with structures that are very similar to those of natural substrates. The decoy molecules force long-alkyl-chain fatty acid hydroxylases (P450BSβ, P450SPα, and P450BM3) to generate the active species and to catalyze oxidation of various substrates other than fatty acids. Interestingly, the catalytic activity was highly dependent on the structure of decoy molecules. Furthermore, the enantioselectivity of reactions catalyzed by P450BSβ and P450SPα was also dependent on the structure of decoy molecules. The decoy molecule system allows us to control reactions catalyzed by wild-type enzymes by designing decoy molecules. [ABSTRACT FROM AUTHOR]

P450 enzymes (P450s) are well known for their ability to oxidize unactivated C H bonds with high regio- and stereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts. Although bacterial P450s typically show higher activity than their mammalian counterparts, they tend to be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkable substrate promiscuity, yet product prediction remains challenging. Protein engineering is one established strategy to overcome these issues. A less explored, yet promising alternative involves substrate engineering. This review discusses the use of small molecules for controlling the substrate specificity and product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalent decoy molecules, and the other involving covalent substrate modifications. [ABSTRACT FROM AUTHOR]

Playing tricks on enzymes: Direct hydroxylation of benzene to phenol was catalyzed by wild‐type P450BM3 in the presence of perfluorinated carboxylic acids as decoy molecules. The catalytic turnover rate reached 120 min−1 per P450. The selectivity towards phenol production was very high and no overoxidation products were detected. [ABSTRACT FROM AUTHOR]

This review surveys our recent studies on artificial heme oxygenases, which consists of two topics. The first topic is an artificial peroxidase founded on a thermally tolerant protein, which shows high thermal stability in the catalytic reaction. In the second topic, we describe 'Decoy system' that has been developed to transform Cytochrome P450 into a versatile oxygenase. [ABSTRACT FROM AUTHOR]