Analysis of critical residues for peroxygenation and improved peroxygenase activity via in situ H2O2 generation in CYP105D18

Bashu Dev Pardhe; Tae-Jin Oh

doi:10.3389/fmicb.2023.1296202

Analysis of critical residues for peroxygenation and improved peroxygenase activity via in situ H₂O₂ generation in CYP105D18

Front Microbiol. 2023 Dec 11:14:1296202. doi: 10.3389/fmicb.2023.1296202. eCollection 2023.

Authors

Bashu Dev Pardhe¹, Tae-Jin Oh^{1

2

3}

Affiliations

¹ Department of Life Science and Biochemical Engineering, Graduate School, SunMoon University, Asan, Republic of Korea.
² Genome-Based BioIT Convergence Institute, Asan, Republic of Korea.
³ Department of Pharmaceutical Engineering and Biotechnology, SunMoon University, Asan, Republic of Korea.

Abstract

Limited numbers of CYPs have been reported to work naturally as peroxygenases. The peroxide shunt pathway can be efficiently used as an alternative for the NAD(P)H and reductase systems, particularly in high hydrogen peroxide (H₂O₂) resistance CYPs. We reported the structural and biochemical features of CYP105D18 peroxygenase for its high H₂O₂ tolerance capacity. Q348 was a crucial residue for the stability of CYP105D18 during the exposure to H₂O₂. In addition, the role of the hydrophilic amino acid T239 from the I helix for peroxygenation and regiospecificity toward testosterone was investigated. Interestingly, T239E differs in product formation from wild type, catalyzing testosterone to androstenedione in the presence of H₂O₂. The other variant, T239A, worked with the Pdx/Pdr system and was unable to catalyze testosterone conversion in the presence of H₂O₂, suggesting the transformation of peroxygenase into monooxygenase. CYP105D18 supported the alternative method of H₂O₂ used for the catalysis of testosterone. The use of the same concentration of urea hydrogen peroxide adducts in place of direct H₂O₂ was more efficient for 2β-hydroxytestosterone conversion. Furthermore, in situ H₂O₂ generation using GOx/glucose system enhanced the catalytic efficiency (k_cat/K_m) for wild type and F184A by 1.3- and 1.9-fold, respectively, compared to direct use of H₂O₂ The engineering of CYP105D18, its improved peroxygenase activity, and alteration in the product oxidation facilitate CYP105D18 as a potential candidate for biotechnological applications.

Keywords: CYP105D18; glucose oxidase; mutagenesis; peroxygenase activity; testosterone.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by the project entitled “Development of potential antibiotic compounds using polar organism resources (20200610),” funded by the Ministry of Oceans and Fisheries, Korea.