Anaerobic Biodegradation of Steroids

Chiang, Yin-Ru; Ismail, Wael

doi:10.1007/978-3-319-33598-8_9-1

Cited by 8 publications

(7 citation statements)

References 144 publications

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“…However, subsequent research demonstrated a high affinity of the enzyme for C19 – C21 steroids and indicated that AcmB is located on the cytoplasmic side of the inner membranes of S. denitrificans [ 8 , 27 ] . These results suggested that androst-4-en-3,17-dione (AD; 1 ) may be the enzyme native substrate [ 28 ]. Nevertheless, AcmB’s ability to catalyze Δ 1 -dehydrogenation of steroids with the aliphatic side chain on the C17 position seemed interesting and quite unusual.…”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…These results suggested that androst-4-en-3,17-dione (AD; 1) may be the enzyme native substrate [27]. Nevertheless, AcmB's ability to catalyze Δ 1 -dehydrogenation of steroids with the aliphatic side chain on the C17 position seemed interesting and quite unusual.…”

Section: Introductionmentioning

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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“…We did not detect this particular 25-hydroxysteroid intermediate in SCN samples; instead, homologues of the C25DH molybdopterin-containing subunits (S25dA1–A7) were identified, suggesting the occurrence of anaerobic 25-hydroxylation in the SCN samples. The hydrophilic CoA-ester intermediates were absent during side chain degradation, and this was partially due to the ethyl acetate extraction procedure and poor ionization of these hydrophilic metabolites in the APCI mode used in this study ( 32 ). Instead, we identified C 26 and C 22 carboxylic intermediates and ACS responsible for the activation of C 24 and C 22 , indicating the degradation of cholesterol side chain through β-oxidation reactions in anoxic sludge communities.…”

Section: Discussionmentioning

confidence: 94%

Microbial Functional Responses to Cholesterol Catabolism in Denitrifying Sludge

Wei

Lee

et al. 2018

mSystems

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Steroids are ubiquitous and abundant natural compounds that display recalcitrance. Biodegradation via sludge communities in wastewater treatment plants is the primary removal process for steroids. To date, compared to studies for aerobic steroid degradation, the knowledge of anaerobic degradation of steroids has been based on only a few model organisms. Due to the increase of anthropogenic impacts, steroid inputs may affect microbial diversity and functioning in ecosystems. Here, we first investigated microbial functional responses to cholesterol, the most abundant steroid in sludge, at the community level. Our metagenomic and metatranscriptomic analyses revealed that the capacities for cholesterol approach, uptake, and degradation are unique traits of certain low-abundance betaproteobacteria, indicating the importance of the rare biosphere in bioremediation. Apparent expression of genes involved in cofactor de novo synthesis and salvage pathways suggests that these micronutrients play important roles for cholesterol degradation in sludge communities.

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Anaerobic Biodegradation of Steroids

Cited by 8 publications

References 144 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

Microbial Functional Responses to Cholesterol Catabolism in Denitrifying Sludge

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