Related Experiment Video
Updated: Sep 19, 2026

CRISPR-Mediated Reorganization of Chromatin Loop Structure
Published on: September 14, 2018
Effect of Linker Sequence on the Interdomain Dynamics of Self-Sufficient CYP116B5-SOX Chimeras
Daniele Giuriato1, Lorenzo Gorga1, Silvia Castrignanò1
1Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, Torino 10123, Italy.
Abstract:
In this work, the design of CYP116B5-SOX-(PPII) is presented. This is an artificial self-sufficient enzyme in which the P450 116B5 peroxygenase and the H2O2-generating sarcosine oxidase (SOX) are linked together in a fusion complex. A polyproline type II helix (PPII) linker was used to connect the two enzymes, obtaining a rigid fusion protein. CYP116B5-SOX-(PPII) displayed 10-fold increased activity in terms of p-nitrophenol oxidation (k cat: 32.1 ± 0.5 min-1; K M: 150 ± 15 μM) compared to the isolated heme domain CYP116B5-hd, and more than 2-fold higher activity compared to CYP116B5-SOXa fusion enzyme in which the two domains are passively linked by a flexible polyglycine sequence. The stability of the three CYP116B5 variants was investigated by differential scanning calorimetry. The data revealed the higher folding cooperativity of the P450 domain within the rigid fusion system, as well as the stabilization of the SOX domain compared to CYP116B5-SOX. The catalytic stability of the fusion enzymes was confirmed by testing the single domains' residual activity after thermal denaturation. The interdomain plasticity of the two fusion enzymes was analyzed by H/D exchange kinetics experiments through ATR-FTIR spectroscopy. The results revealed that the flexible CYP116B5-SOX exhibits a globally lower protein surface accessibility to water compared to the rigid CYP116B5-SOX-(PPII), suggesting that the formation of interdomain contacts is disadvantageous for this fusion couple. CYP116B5 shows some activity toward styrene oxidation into styrene oxide and phenylacetaldehyde (K M: 9.0 ± 2.9 mM, k cat: 1.9 ± 0.3 min-1). Interestingly, E. coli cells expressing CYP116B5-SOX-(PPII) show 56% styrene conversion when hydrogen peroxide is produced in situ by SOX, whereas only 13% conversion is reached when hydrogen peroxide is directly added in solution. Overall, this work shows that the stability and catalytic efficiency of the artificial chimera are intimately associated with the quaternary assembly of the construct, unveiling the crucial role of the fusion design for the extensive exploitation of P450 biocatalysis.
Related Concept Videos
Protein Complexes with Interchangeable Parts
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...
Conserved Binding Sites
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Exon Recombination
Exon shuffling follows “splice frame rules.” Each exon has three reading...
Cooperative Binding of Transcription Regulators
