Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques

Sofińska, Kamila; Wojtkiewicz, Agnieszka M.; Wójcik, Patrycja; Zastawny, Olga; Guzik, Maciej; Winiarska, Agnieszka; Waligórski, Piotr; Cieśla, Michał; Barbasz, Jakub; Szaleniec, Maciej

doi:10.1016/j.bbagen.2019.03.009

Cited by 12 publications

(31 citation statements)

References 74 publications

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“…The homology modeling of KSTD from Arthrobacter simplex (active towards cholest-4-en-3-one) showed the presence of an additional loop close to the enzyme active site, non-observed in the crystal structure of KSTD1 form R. erythropolis [ 34 , 51 ]. Interestingly, the same feature was noticed in the AcmB homology model [ 52 ].…”

Section: Discussionsupporting

confidence: 66%

“…Nevertheless, the affinity and the catalytic efficiency of KSTD1 from R. erythropolis for cholest-4-en-3-one turned out to be significantly lower than that determined for AcmB from S. denitrificans . Additionally, a more hydrophobic character of the AcmB surface when compared to KSTD1 may also contribute to its higher affinity to more hydrophobic substrates [ 47 ].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionsupporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…Nevertheless, the affinity and the catalytic efficiency of KSTD1 from R. erythropolis for cholest-4-en-3-one turned out to be significantly lower than that determined for AcmB from S. denitrificans. Additionally, a more hydrophobic character of the AcmB surface when compared to KSTD1 may also contribute to its higher affinity to more hydrophobic substrates [46].…”

Section: Discussionmentioning

confidence: 99%

Universal Capability of 3-Ketosteroid Δ1-Dehydrogenases to Catalyze Δ1-Dehydrogenation of C17- Substituted Steroids

Wójcik

Glanowski

Wojtkiewicz

et al. 2021

Preprint

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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.

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“…The protein was obtained in heterologous overexpression in E. coli BL21(DE3)Magic with cloned pMCSG7 vector containing acmb gene from S. denitrificans (gi: 157673245) [15].…”

Section: Acmb Production and Purificationmentioning

confidence: 99%

“…However, after 48 h under reaction conditions, the activity of the homogenous enzyme was 8.5 lower than the activity of the immobilized catalyst. The most probable reason, besides a standard thermal denaturation of the protein that proceeds faster for non-immobilized enzymes, is a gradual aggregation of the enzyme at the optimal pH of the reaction (pH 6.5)[15]. This aggregation is completely inhibited by the immobilization.As a result, it was possible to run an efficient long-term fed-batch reactor with immobilized AcmB at MCF/GA.…”

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confidence: 99%

Application of Immobilized Cholest-4-en-3-one Δ<sup>1</sup>-Dehydrogenase from <em>Sterolibacterium denitrificans</em> for Dehydrogenation of Steroids

Tataruch¹,

Wójcik²,

Wojtkiewicz³

et al. 2020

Preprint

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Cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans is successfully immobilized on 3-aminopropyltrimethoysilane functionalized MCF and SBA-15 silica supports using adsorption or covalently with glutaraldehyde or divinyl sulfone linkers. The best catalyst, AcmB on MCF linked covalently with glutaraldehyde, retains the specific activity of the homogenous enzyme while exhibiting a substantial increase of the operational stability. The immobilized enzyme was used continuously in the fed-batch reactor for 27 days, catalyzing 1,2-dehydrogenation of androst-4-en-3-one to androst-1,4-dien-3-one with a final yield of 29.9 mM (8.56 g/L) and 99% conversion. The possibility of reuse of the immobilized catalyst was also demonstrated and resulted with a doubling of the product amount compared to that in the reference homogenous reactor. Finally, it was shown that molecular oxygen from the air can efficiently be used as an electron acceptor either reoxidizing directly the enzyme or the reduced DCPIPH2. Keywords: 3-ketosteroid D1-dehydrogenase; KSTD; KSDH; AcmB; 1,2-dehydrogenation; cholest-4-en-3-one Δ1-dehydrogenase; enzyme immobilization, FAD-dependent enzymes; enzyme immobilization;

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Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques

Cited by 12 publications

References 74 publications

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

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

Universal Capability of 3-Ketosteroid Δ1-Dehydrogenases to Catalyze Δ1-Dehydrogenation of C17- Substituted Steroids

Application of Immobilized Cholest-4-en-3-one Δ<sup>1</sup>-Dehydrogenase from <em>Sterolibacterium denitrificans</em> for Dehydrogenation of Steroids

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