Enzymatic synthesis of a skin active ingredient - glochidone by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans

Agnieszka M Wojtkiewicz; Gabriela Oleksy; Magdalena A Malinowska; Tomasz Janeczko

doi:10.1016/j.jsbmb.2024.106513

Enzymatic synthesis of a skin active ingredient - glochidone by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans

J Steroid Biochem Mol Biol. 2024 Mar 21:241:106513. doi: 10.1016/j.jsbmb.2024.106513. Online ahead of print.

Authors

Agnieszka M Wojtkiewicz¹, Gabriela Oleksy², Magdalena A Malinowska³, Tomasz Janeczko⁴

Affiliations

¹ Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, Krakow PL30239, Poland. Electronic address: agnieszka.wojtkiewicz@ikifp.edu.pl.
² Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, Krakow PL30239, Poland.
³ Organic Chemistry and Technology Department, Faculty of Chemical Engineering and Technology, Cracow University of Technology, Warszawa 24, Krakow 31-155, Poland.
⁴ Department of Food Chemistry and Biocatalysis, Wrocław University of Environmental and Life Sciences, Norwida 25, Wrocław 50-375, Poland.

PMID: 38521362
DOI: 10.1016/j.jsbmb.2024.106513

Abstract

In this study, we applied AcmB2, sourced from Sterolibacterium denitrificans, to catalyze the oxidative dehydrogenation of 3-ketolupeol (lupenone), a derivative of lupeol, triterpene obtained from birch bark. This enzymatic Δ¹-dehydrogenation catalyzed by AcmB2 yielded glochidone, a bioactive compound frequently obtained from medicinal plants like Salvia trichoclada and Maytenus boria. Glochidone is known for its broad biological activities, including antibacterial, antifungal, anti-inflammatory, anticancer, antidiabetic as well as acetylcholinesterase inhibition. Our research demonstrates >99% conversion efficiency with 100% regioselectivity of the reaction. The effective conversion to glochidone employed an electron acceptor e.g., potassium hexacyanoferrate III, in mild, environmentally friendly conditions: 8-16% 2-hydroxypropyl-β-cyclodextrin, and 2-3% 2-methoxyethanol. AcmB2 reaction optimum was determined at pH 8.0 and 30 °C. Enzyme's biochemical attributes such as electron acceptor type, concentration and steroid substrate specificity were investigated. Among 4-, 5- and 6-ring steroid derivatives androst-4-en-3,17-dione and testosterone propionate were determined as the best substrates of AcmB2. Δ¹-Dehydrogenation of substrates such as lupenone, diosgenone and 3-ketopetromyzonol was confirmed. We have assessed the antioxidant and rejuvenating characteristics of glochidone as an active component in formulations, considering its precursors, lupeol, and lupenone as well. Glochidone exhibited limited antioxidant and chelating capabilities compared to lupeol and reference compounds. However, it demonstrated robust rejuvenating properties, with a sirtuin induction level of 61.5 ± 1.87%, notably surpassing that of the reference substance, E-resveratrol (45.15 ± 0.09%). Additionally, glochidone displayed 26.5±0.67 and 19.41±0.76% inhibition of elastase and collagenase, respectively. The safety of all studied triterpenes was confirmed on skin reconstructed human Epidermis model. These findings provide valuable insights into the potential applications of glochidone in formulations aimed at addressing skin health concerns. This research presents the first example of an enzyme in the 3-ketosteroid dehydrogenase (KstD) family catalyzing the Δ¹-dehydrogenation of a pentacyclic triterpene. We also explored structural differences between AcmB, AcmB2, and related KstDs pointing to G52 and P532 as potentially responsible for the unique substrate specificity of AcmB2. Our findings not only highlight the enzyme's capabilities but also present novel enzymatic pathways for bioactive compound synthesis.

Keywords: 1-dehydro-lupenone; 3-ketosteroid dehydrogenase; Antiaging activity; Cholest-4-en-3-one dehydrogenase 2; Glochidone; Pentacyclic triterpenoids; Sterolibacterium denitrificans.