Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids

Abstract

Background 3-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. Results Molecular 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). Conclusions 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.

Structure, mutagenesis and QM:MM modelling of 3-ketosteroid Δ1-dehydrogenase from Sterolibacterium denitrificans – the role of new membrane-associated domain and proton-relay system in catalysis

“…Theoretical predictions of ∆Gb obtained for AcmB turned out to be similar to those published for KstD1 40 (Table S4-S6). The estimated free energy of binding for the effective enzyme-substrate complexes (G) for all analysed substrates ranged between -10 and -3.4 kcal/mol (Table S4), but the Gs of the substrates with a degraded C17 substituent were in the range of -7.6 to -3.7 kcal/mol.…”

“…Cholest-4-en-3-one was not different from other substrates (Gb of -6.3 kcal/mol), but diosgenon exhibited better stabilization than for AcmB (-5.8 kcal/mol). These results confirm the preferential binding of cholest-4-en-3-one by the enzyme with an additional 'loop' and explain the better apparent activity of KstD1 with diosgenone compared to AcmB 40 . This result also suggests that the membrane-associated domain plays a role in the differentiation of substrates with extended C17 substituent (i.e., preferential binding of CHON over DSG).…”

“…Theoretical prediction indicates that such anchoring positions the enzyme active site oriented toward the membrane surface, which may facilitate the formation of the enzyme-substrate complex even with highly hydrophobic steroids. This domain also narrows the entrance to the active site and strongly interacts with the extended alkyl sidechain in cholest-4-en-3-one, a native substrate of AcmB 40 . Prediction of Gbinding and the energy of interaction supports this hypothesis, especially compared to the results calculated for KstD1, which lacks preference for binding cholest-4-en-3-one over steroids with degraded C17 substituent.…”

“…From each trajectory, two ∆Gb estimates were received: total ∆Gb for the whole simulation and ∆Gbest estimated for the five nanoseconds simulation with the best geometry parameters of the bound substrate. These results were compared with MM-PBSA analysis of enzyme: substrate MD simulations of KstD1 from R. erythropolis 40 , which were analyzed according to the above protocol.…”

“…Recently we have reported that kinetics of AcmB point to cholest-4-en-3-one (CHON) as a native substrate 40 . AcmB differs from KstD1 by the presence of a 40 amino acid fragment (153-204) that was formerly referred to as a 'loop' but that in fact forms a membrane-associated domain at the entrance of the active site (Figures 3E and F).…”