ACS Omega. 2026 Sep 1;11(36):54593-54602. doi: 10.1021/acsomega.6c07054. eCollection 2026 Sep 15.

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-SOXa 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.

PMID:42756812 | PMC:PMC13584785 | DOI:10.1021/acsomega.6c07054