Work

A second-sphere variant of cytochrome P450 PbdA improves turnover and apparent coupling in veratrate oxidation

Year: 2026

Type: article

Abstract: Efficient use of reducing equivalents is critical for productive turnover in cytochrome P450 redox enzymes, yet improving activity without compromising substrate affinity remains challenging. Here, we engineered the second-sphere region of PbdA, a cytochrome P450 from Rhodococcus jostii RHA1 that catalyzes veratrate oxidation, and identified position 242 as a functionally sensitive site. The hydrophobic D242V variant approximately doubled the catalytic turnover number, k cat , while largely preserving substrate binding affinity. By quantifying NADH consumption and product formation, we found that D242V increased apparent coupling efficiency from 43.88% to 57.30%. Molecular dynamics simulations suggested that V242 remodels active-site hydration, reducing local water occupancy, which may help limit nonproductive uncoupling. Pathways analysis further revealed modest differences in the sampling of putative electron-transfer geometries, which correlated with the observed kinetic improvement but should be interpreted as a relative structural descriptor rather than direct evidence for altered electron-transfer rates. Finally, coupling engineered PbdA with glyoxylate carboligase enabled in situ formaldehyde scavenging and glycolaldehyde synthesis, achieving approximately 90% substrate conversion and 70% glycolaldehyde yield. These results suggest that while the second-sphere generally accommodates a highly restrictive mutational landscape, precise substitutions at isolated sensitive nodes (such as position 242) can occasionally fine-tune turnover and apparent coupling in P450-catalyzed veratrate oxidation, providing a basis for related biocatalytic cascades.

Source: Bioresources and Bioprocessing

DOI: 10.1186/s40643-026-01130-4

Authors: Haiyan Song, Zeyang Li, Andong Li, Xinyu Cui, Shangxian Xie, Zhiguang Zhu

Institutions: Chinese Academy of Sciences, Tianjin Institute of Industrial Biotechnology, Huazhong University of Science and Technology, University of Chinese Academy of Sciences

Language: English

Topic: Enzyme Catalysis and Immobilization

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,890

Funders: National Natural Science Foundation of China, National Key Research and Development Program of China, Science Fund for Distinguished Young Scholars of Tianjin