Regioselective hydroxylation of cholecalciferol, cholesterol and other sterol derivatives by steroid C25 dehydrogenase

Abstract

Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6-9 % (w/v) 2-hydroxypropyl-β-cyclodextrin as a solubilizer and 1.25-5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8-15 mM K[Fe(CN)] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min mg), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms

“…S25DH is a molybdenum enzyme that belongs to the so-called EBDH-like hydroxylases (Dermer and Fuchs 2012 ; Heider et al 2016 ), where EBDH stands for ethylbenzene dehydrogenase, the first enzyme of the class, (Heider et al 2016 ; Kniemeyer and Heider 2001 ). Similarly to EBDH, S25DH enables a highly regioselective hydroxylation of unactivated hydrocarbons, i.e., various steroids, exclusively at the C25 atom utilizing an oxygen atom from water instead of molecular oxygen (Chiang et al 2007 ; Rugor et al 2017a ; Szaleniec et al 2014 ).…”

“…The potential application of S25DH for biocatalytic synthesis of calcifediol seems to be especially attractive since recent establishment of the overexpression system as recombinant S25DH1 preparation is 6.5-fold more active than wild-type extract and enables conversion of 0.38 g/L of VD3 in 3 h (Jacoby et al 2018 ). The proposed system was also applied for hydroxylation of various 3-ketosteroids such as: cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one with a final concentration in the range of 2.2 g/L, or sterols such as: cholesterol, 7-dehydrocholesterol, with a final concentration in the range of 0.8 g/L (Rugor et al 2017a ). Purified S25DH was also efficiently immobilized on silica (Rugor et al 2017a ) or on a microfiltration membrane (Kalimuthu et al 2018 ).…”

“…However, the enzyme/s involved in this 6-hydroxylation of testosterone are still unknown (Wang et al 2014 ). To date, only for S25DH, the biotechnological applications have been established for the hydroxylation of the cholesterol derivate and, most importantly, the production of 25-hydroxy vitamin D 3 (calcifediol or calcidiol) (Rugor et al 2017a ).…”

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

“…For example, when proteins such as S25DH are assembled and loaded with a redox cofactor within the cytoplasm, they must be transported through the membrane in a fully folded form to the dedicated periplasmic space [72], which involves tight regulations by specific chaperones for cofactor insertion [15]. Although the protocol for purification of the steroid C25 dehydrogenase from its natural source has been already established [27] and simplified [35], the successful recombinant expression of the S25DH would have a significant impact on the production of sterol-based pharmaceutics. To tackle the multi-subunit, multi-cofactors issue we have adopted a “divide-and-conquer” strategy [73] – the main goal of obtaining the protein with a cofactor was divided into smaller independent steps that were separately solved, leading to the answer of how to obtain the cofactor-loaded chaperone.…”

“…calciferol) [13,35] to the respective tertiary alcohol [27]. S25DH is a heterotrimer with αβγ composition that belongs to the DMSO reductase family.…”