Universal capability of 3-ketosteroid [DELA]1-dehydrogenases to catalyze [DELA]1-dehydrogenation of C17-substituted steroids

• Published in: Microbial cell factories Vol. 20; no. 1
• Main Authors: Wójcik, Patrycja, Glanowski, MichaÅ, Wojtkiewicz, Agnieszka M, Rohman, Ali, Szaleniec, Maciej
• Format: Journal Article
• Language: English
• Published: BioMed Central Ltd 23-06-2021
• Subjects: Chemical properties; Chemical research; Dehydrogenation; Enzyme kinetics; Microbial enzymes; Oxidoreductases; Steroids; Substrates (Biochemistry); Poland
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-021-01611-5

3-Ketosteroid [DELA].sup.1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD's substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-[beta]-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in [DELA]G.sub.bind between the native substrate, androst-4-en-3,17-dione (AD; - 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (- 8.40 kcal/mol) or diosgenone (- 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi K.sub.mA = 23.7 [mu]M) than to AD (K.sub.mA = 529.2 [mu]M), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (k.sub.cat/K.sub.mA = 9.25*10.sup.6 M.sup.-1 s.sup.-1) is almost 20 times higher than measured for KSTD1 (k.sub.cat/K.sub.mA = 4.71*10.sup.5 M.sup.-1 s.sup.-1). We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.