Tunnel engineering for modulating the substrate preference in cytochrome P450BsβHI

Abstract

An active site is normally located inside enzymes, hence substrates should go through a tunnel to access the active site. Tunnel engineering is a powerful strategy for refining the catalytic properties of enzymes. Here, P450BsβHI (Q85H/V170I) derived from hydroxylase P450Bsβ from Bacillus subtilis was chosen as the study model, which is reported as a potential decarboxylase. However, this enzyme showed low decarboxylase activity towards long-chain fatty acids. Here, a tunnel engineering campaign was performed for modulating the substrate preference and improving the decarboxylation activity of P450BsβHI. The finally obtained BsβHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; BsβHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. The study demonstrates how the substrate preference can be modulated by tunnel engineering strategy.

Engineered Stable 5-Hydroxymethylfurfural Oxidase (HMFO) from 8BxHMFO Variant of Methylovorus sp. MP688 through B-Factor Analysis

“…Compared to others, Q319K shows better performance under each of the reaction temperatures, and from the tendency of FDCA yields, it could be concluded that the improved HMFO mutants have better yields with shorter reaction time than the original 8BxHMFO within a temperature range of 30 to 40 • C. It is interesting that the catalytic result with combinatorial mutation N44G-Q319K were worse than the two of single site mutants; the reason for this may be that the combinatorial mutation influenced the structure of active site and thus decreased the catalytic performance. Similar results were also proved in other works of enzyme evolution [23]. The performance of each mutant with higher substrate concentration was also investigated.…”

Flexibility Regulation of Loops Surrounding the Tunnel Entrance in Cytochrome P450 Enhanced Substrate Access Substantially

“…5 A bottleneck is the narrowest point among tunnel profiles, which is the general research hotspot in tunnel engineering. 2,6 For instance, Cui et al revealed that the CYP2E1 tunnel bottleneck residues (H107, A108, and H109) could affect the ligand affinity with the substrate (arachidonic acid) through electrostatic interactions. 7 In addition, Prakinee et al reduced the halogenating intermediate leakage from the active sites of tryptophan 6halogenase through engineered V82 residue on the tunnel bottleneck, which connects the two active sites of flavindependent halogenases.…”

“…17 In studying the loops and the tunnels, cytochrome P450s could be one of the typical models used for systematic research. 2,13,17,18 The cytochrome P450 (CYPs), a large family of heme enzymes, is widely distributed in nature and one of the most universal biological catalysts. 19−21 These enzymes can catalyze a variety of reactions, such as dealkylation reactions, hydroxylation, epoxidation, deamination, C−C and C−O coupling reactions, and so on.…”

In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators

“…Docking helps to understand the molecular interaction between ligands and receptors and can therefore be used for further binding improvement through ligand modification (drugs) or receptor modification (protein engineering). In addition to that it can also be used for molecular characterization of already engineered proteins [25] or for characterization of protein-protein interactions [26]. Nearly all docking programs and the advantages and disadvantages of docking are well summarized and explained more in detail in [27].…”