Abstract
Background3-Ketosteroid Δ1-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-β-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.ResultsMolecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind 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 KmA = 23.7 μM) than to AD (KmA = 529.2 μM), 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 (kcat/KmA = 9.25∙106 M−1 s−1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M−1 s−1).ConclusionsWe 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.
Highlights
Steroids are widespread compounds in the natural environment
The Molecular dynamics (MD) simulation of ketosteroid Δ1-dehydrogenase from R.erythropolis (KSTD1):cholest-4-en-3-one complex revealed that the enzyme binds the steroid ring core while the isooctyl substituent at C17 position protrudes out of the active site and generally does not prevent it from the active binding (Fig. 2)
There was no difference in ΔGbind between the native substrate of KSTD1 from R. erythropolis, androst-4-en-3,17-dione (− 8.02 ± 0.13 kcal/ mol, 1), and cholest-4-en-3-one (− 8.40 ± 0.19 kcal/mol, 2)
Summary
Steroids are widespread compounds in the natural environment. They are essential components of eukaryotic i.e., animal, yeast and fungi and plant, cells. Cholesterol, the parent substance of all sterols, is one of the most. The microbial degradation of cholesterol proceeds under either aerobic or anaerobic conditions and the current stage of knowledge was recently reviewed by Rohman et al [2]. 3-Ketosteroid Δ1-dehydrogenases (KSTDs) initiate the crucial step of the degradation – steroid nucleus decomposition and as a result are found in aerobic, facultative anaerobic and strictly anaerobic microorganisms [3,4,5]. The stage at which the double bond is introduced during cholesterol degradation is still debated and may differ between bacterial species [2]
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