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Universal capability of 3-ketosteroid Δ(1)-dehydrogenases to catalyze Δ(1)-dehydrogenation of C17-substituted steroids

BACKGROUND: 3-Ketosteroid Δ(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 tho...

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Autores principales: Wójcik, Patrycja, Glanowski, Michał, Wojtkiewicz, Agnieszka M., Rohman, Ali, Szaleniec, Maciej
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8220720/
https://www.ncbi.nlm.nih.gov/pubmed/34162386
http://dx.doi.org/10.1186/s12934-021-01611-5
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author Wójcik, Patrycja
Glanowski, Michał
Wojtkiewicz, Agnieszka M.
Rohman, Ali
Szaleniec, Maciej
author_facet Wójcik, Patrycja
Glanowski, Michał
Wojtkiewicz, Agnieszka M.
Rohman, Ali
Szaleniec, Maciej
author_sort Wójcik, Patrycja
collection PubMed
description BACKGROUND: 3-Ketosteroid Δ(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-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 ΔG(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(mA) = 23.7 μM) than to AD (K(mA) = 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 (k(cat)/K(mA) = 9.25∙10(6) M(−1) s(−1)) is almost 20 times higher than measured for KSTD1 (k(cat)/K(mA) = 4.71∙10(5) 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01611-5.
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spelling pubmed-82207202021-06-23 Universal capability of 3-ketosteroid Δ(1)-dehydrogenases to catalyze Δ(1)-dehydrogenation of C17-substituted steroids Wójcik, Patrycja Glanowski, Michał Wojtkiewicz, Agnieszka M. Rohman, Ali Szaleniec, Maciej Microb Cell Fact Research BACKGROUND: 3-Ketosteroid Δ(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-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 ΔG(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(mA) = 23.7 μM) than to AD (K(mA) = 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 (k(cat)/K(mA) = 9.25∙10(6) M(−1) s(−1)) is almost 20 times higher than measured for KSTD1 (k(cat)/K(mA) = 4.71∙10(5) 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01611-5. BioMed Central 2021-06-23 /pmc/articles/PMC8220720/ /pubmed/34162386 http://dx.doi.org/10.1186/s12934-021-01611-5 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Wójcik, Patrycja
Glanowski, Michał
Wojtkiewicz, Agnieszka M.
Rohman, Ali
Szaleniec, Maciej
Universal capability of 3-ketosteroid Δ(1)-dehydrogenases to catalyze Δ(1)-dehydrogenation of C17-substituted steroids
title Universal capability of 3-ketosteroid Δ(1)-dehydrogenases to catalyze Δ(1)-dehydrogenation of C17-substituted steroids
title_full Universal capability of 3-ketosteroid Δ(1)-dehydrogenases to catalyze Δ(1)-dehydrogenation of C17-substituted steroids
title_fullStr Universal capability of 3-ketosteroid Δ(1)-dehydrogenases to catalyze Δ(1)-dehydrogenation of C17-substituted steroids
title_full_unstemmed Universal capability of 3-ketosteroid Δ(1)-dehydrogenases to catalyze Δ(1)-dehydrogenation of C17-substituted steroids
title_short Universal capability of 3-ketosteroid Δ(1)-dehydrogenases to catalyze Δ(1)-dehydrogenation of C17-substituted steroids
title_sort universal capability of 3-ketosteroid δ(1)-dehydrogenases to catalyze δ(1)-dehydrogenation of c17-substituted steroids
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8220720/
https://www.ncbi.nlm.nih.gov/pubmed/34162386
http://dx.doi.org/10.1186/s12934-021-01611-5
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