Using Small Molecules to Enhance P450 OleT Enzyme Activity in Situ

Abstract

Cytochrome P450 OleT is a fatty acid decarboxylase that catalyzes the production of olefins with biofuel and synthetic applications. However, the relatively sluggish catalytic efficiency of the enzyme limits its applications. Here, we report the application of a novel class of benzene containing small molecules to improve the OleT activity. The UV-Vis spectroscopy study and molecular docking results confirmed the high proximity of the small molecules to the heme group of OleT. Up to 6-fold increase of product yield has been achieved in the small molecule-modulated enzymatic reactions. Our work thus sheds the light to the application of small molecules to increase the OleT catalytic efficiency, which could be potentially used for future olefin productions.

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

“…Not like the DFSM system which assists to activate H 2 O 2 in the H 2 O 2 ‐innert P450BM3 variant, [25] this class of small molecules worked on wild type peroxygenase OleT. The authors proposed that the small molecules lead to more efficiently utilization of H 2 O 2 in the enzymatic reaction, meanwhile released the oxidative stress of H 2 O 2 , which was concluded based on a H 2 O 2 titration assay and a glucose oxidase (GOx) related H 2 O 2 releasing assay [82,75] . The limitations of the system was that the small molecules showed varied effect on different substrates.…”

“…Very recently, Wang group reported a class of small molecules that could significantly enhance OleT activity in situ. Up to 6‐fold increase of product yields has been achieved [75] . Inspired by the phenomenon that the fine‐tuned substrate structure could influence the enzymatic outcome, [45] and the perdeuterated fatty acid to trigger OleT Cpd I to oxidize norcarane, [31] Zhang et al .…”

“…[75] Inspired by the phenomenon that the fine-tuned substrate structure could influence the enzymatic outcome, [45] and the perdeuterated fatty acid to trigger OleT Cpd I to oxidize norcarane, [31] Zhang et al proposed the usage of small aromatic molecules to influence the fatty acid coordination, thus to leverage OleT catalysis. [75] They readily synthesized the triazole benzene, sulfonyl amide benzene and other small aromatic molecules (Figure 8C), and revealed that the small molecules could improve the substrate binding to OleT. [75] Not like the DFSM system which assists to activate H 2 O 2 in the H 2 O 2 -innert P450BM3 variant, [25] this class of small molecules worked on wild type peroxygenase OleT.…”

“…[75] They readily synthesized the triazole benzene, sulfonyl amide benzene and other small aromatic molecules (Figure 8C), and revealed that the small molecules could improve the substrate binding to OleT. [75] Not like the DFSM system which assists to activate H 2 O 2 in the H 2 O 2 -innert P450BM3 variant, [25] this class of small molecules worked on wild type peroxygenase OleT. The authors proposed that the small molecules lead to more efficiently utilization of H 2 O 2 in the enzymatic reaction, meanwhile released the oxidative stress of H 2 O 2 , which was concluded based on a H 2 O 2 titration assay and a glucose oxidase (GOx) related H 2 O 2 releasing assay.…”

“…The terminal catalytical group played similar role of the engineered acidbased residual through enzyme mutagenesis, [77] but showed more significant effect on the improvement of enzymatic [65,66,71,72,74] DFSMs (B), [25] and the small molecules used to leverage OleT catalysis (C). [75] Copyright 2021 American Chemical Society. (B) Active site of the P450BM3-F87A mutant in complex with Im-C6-Phe and NH 2 OH (PDB: 7EGN).…”