Characterization of a novel porcine carbonyl reductase activated by glutathione: Relationship to carbonyl reductase 1, 3α/β-hydroxysteroid dehydrogenase and prostaglandin 9-ketoreductase

Satoshi Endo; Yoshifumi Morikawa; Toshiyuki Matsunaga; Akira Hara; Masaki Takasu

doi:10.1016/j.cbi.2023.110572

Characterization of a novel porcine carbonyl reductase activated by glutathione: Relationship to carbonyl reductase 1, 3α/β-hydroxysteroid dehydrogenase and prostaglandin 9-ketoreductase

Chem Biol Interact. 2023 Aug 25:381:110572. doi: 10.1016/j.cbi.2023.110572. Epub 2023 May 27.

Authors

Satoshi Endo¹, Yoshifumi Morikawa², Toshiyuki Matsunaga³, Akira Hara⁴, Masaki Takasu⁵

Affiliations

¹ Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu, 501-1196, Japan; Center for One Medicine Innovative Translational Research (COMIT), Gifu University, Gifu, 501-1193, Japan. Electronic address: sendo@gifu-pu.ac.jp.
² Forensic Science Laboratory, Gifu Prefectural Police Headquarters, Gifu, 500-8501, Japan.
³ Department of Biofunctional Analysis, Gifu Pharmaceutical University, Gifu, 501-1196, Japan.
⁴ Faculty of Engineering, Gifu University, Gifu, 501-1193, Japan.
⁵ Center for One Medicine Innovative Translational Research (COMIT), Gifu University, Gifu, 501-1193, Japan; Institute for Advanced Study, Gifu University, Gifu, 501-1193, Japan.

PMID: 37247810
DOI: 10.1016/j.cbi.2023.110572

Abstract

A porcine gene, LOC100622246, encodes carbonyl reductase [NADPH] 1 (pCBR-N1), whose function remains unknown. Previously, three porcine carbonyl reductases, carbonyl reductase 1 (pCBR1), 3α/β-hydroxysteroid dehydrogenase (p3α/β-HSD) and prostaglandine-9-keto reductase (pPG-9-KR), were purified from neonatal testis, adult testis and adult kidney, respectively. However, the relationship of pCBR-N1 with the three enzymes is still unknown. Here, we compare the properties of the recombinant pCBR-N1 and pCBR1. The two enzymes reduced various carbonyl compounds including 5α-dihydrotestosterone, which was converted to its 3α- and 3β-hydroxy-metabolites. Compared to pCBR1, pCBR-N1 exhibited higher K_m and k_cat values for most substrates, but more efficiently reduced prostaglandin E₂. pCBR-N1 was inhibited by known inhibitors of p3α/β-HSD (hexestrol and indomethacin), but not by pCBR1 inhibitors. pCBR-N1 was highly expressed than pCBR1 in the several tissues of adult domestic and microminiature pigs. The results, together with partial amino acid sequence match between pCBR-N1 and pPG-9-KR, reveal that pCBR-N1 is identical to p3α/β-HSD and pPG-9-KR. Notably, pCBR-N1, but not pCBR1, reduced S-nitrosoglutathione and glutathione-adducts of alkenals including 4-oxo-2-nonenal with K_m of 8.3-32 μM, and its activity toward non-glutathionylated substrates was activated 2- to 9-fold by 1 mM glutathione. Similar activation by glutathione was also observed for human CBR1. Site-directed mutagenesis revealed that the differences in kinetic constants and glutathione-mediated activation between pCBR-N1 and pCBR1 are due to differences in residue 236 and two glutathione-binding residues (at positions 97 and 193), respectively. Thus, pCBR-N1 is a glutathione-activated carbonyl reductase that functions in the metabolism of endogenous and xenobiotic carbonyl compounds.

Keywords: 3α/β-hydroxysteroid dehydrogenase; Activation; CBR1; Glutathione; Prostaglandine-9-keto reductase; S-nitrosoglutathione.

MeSH terms

Alcohol Oxidoreductases* / genetics
Alcohol Oxidoreductases* / metabolism
Animals
Carbonyl Reductase (NADPH)*
Humans
Hydroxyprostaglandin Dehydrogenases / genetics
Hydroxyprostaglandin Dehydrogenases / metabolism
Hydroxysteroid Dehydrogenases / metabolism
Male
Swine

Substances

Alcohol Oxidoreductases
Carbonyl Reductase (NADPH)
Hydroxyprostaglandin Dehydrogenases
Hydroxysteroid Dehydrogenases
prostaglandin-E2 9-reductase