Concerning the Stereochemistry and Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids

Ringold, Howard J.; Hayano, Mika; Stefanovic, Vladimir

doi:10.1016/s0021-9258(18)67926-8

Cited by 60 publications

(13 citation statements)

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“…Such behavior could be explained with an unknown mechanism where both hydrogens are transferred simultaneously, and what would be observed was primary KIE for both substituted positions. This hypothesis was, however, discarded as experimental data, , and results from our calculations show that hydrogen atoms are removed in a step-wise manner. Another potential explanation of the experimental values would be a situation where the barrier heights of TS1 and TS2 were so close, that both of them affected the reaction rate .…”

Section: Results and Discussionmentioning

confidence: 99%

“…17 RHR proceeds in a step-wise manner, that is, the steroid is activated by the tyrosyl anion, which abstracts the acidic 2β-H proton from the C2 atom with a high degree of stereoselectivity. 20 In the second, supposedly rate-limiting step, 1α hydride is shifted to FAD resulting in the formation of the double bond (Figure 1). 14,22 Surprisingly, this hypothesis was never validated with quantum chemical calculations despite several intriguing results which did not entirely agree with the undoubtedly sensible mechanistic hypothesis.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The mechanistic hypothesis describing the catalytic mechanism has been formulated based on (i) the structure of only one KSTD from Rhodococcus erythropolis (PDB 4c3x and 4c3y), , (ii) activity tests of genetically modified enzyme variants, and (iii) numerous kinetic studies that yielded values of the kinetic isotope effect (KIE) for chirally labeled substrates. ,− The currently accepted hypothesis assumes that the overall reaction proceeds according to the Ping-Pong bi–bi mechanism, with the steroid substrate oxidized during reductive half-reaction (RHR) and FAD reoxidation by the electron acceptor during oxidative half-reaction (OHR) . RHR proceeds in a step-wise manner, that is, the steroid is activated by the tyrosyl anion, which abstracts the acidic 2β-H proton from the C2 atom with a high degree of stereoselectivity . In the second, supposedly rate-limiting step, 1α hydride is shifted to FAD resulting in the formation of the double bond (Figure ).…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic Δ¹-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

et al. 2021

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Δ 1 -Dehydrogenation of 3-ketosteroids catalyzed by flavin adenine dinucleotide (FAD)-dependent 3-ketosteroid dehydrogenases (Δ 1 -KSTD) is a crucial step in steroid degradation and synthesis of several steroid drugs. The catalytic mechanism assumes the formation of a double bond in two steps, proton abstraction by tyrosyl ion, and a rate-limiting hydride transfer to FAD. This hypothesis was never verified by quantum-mechanical studies despite contradictory results from the kinetic isotope effect (KIE) reported in 1960 by Jerussi and Ringold [Biochemistry 1965, 4 (10)]. In this paper, we present results that reconcile the mechanistic hypothesis with experimental evidence. Quantum mechanics/molecular mechanics molecular dynamics simulations show that the proposed mechanism is indeed the most probable, but barriers associated with substrate activation (13.4−16.3 kcal•mol −1 ) and hydride transfer (15.5−18.0 kcal•mol −1 ) are very close (1.7−2.1 kcal•mol −1 ), which explains normal KIE values for steroids labeled either at C1 or C2 atoms. We confirm that tyrosyl ion acting as the catalytic base is indeed necessary for efficient activation of the steroid. We explain the lower value of the observed KIE (1.5−3.5) by the nature of the free energy surface, the presence of diffusion limitation, and to a smaller extent, conformational changes of the enzyme upon substrate binding. Finally, we confirm the Ping-Pong bi−bi kinetics of the whole Δ 1 -dehydrogenation and demonstrate that substrate binding, steroid dehydrogenation, and enzyme reoxidation proceed at comparable rates.

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Section: Results and Discussionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Enzymatic Δ¹-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

et al. 2021

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“…A sample of this material which was dehydrogenated by a cell-free preparation of B. sphaericus in the presence of menadione by the procedure previously described (Ringold et al, 1963) exhibited an 86% loss of deuterium.…”

Section: Preparation Of Deuterated Steroidsmentioning

confidence: 99%

The Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids. III. Kinetics and Isotope Effects^*

Jerussi¹,

Ringold²

1965

Biochemistry

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“…KSTDs catalyze regio- and stereoselective dehydrogenation between C1 and C2 atoms of steroid ring A (Fig. 1 ) [ 6 ]. The stage at which the double bond is introduced during cholesterol degradation is still debated and may differ between bacterial species [ 2 ].…”

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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Concerning the Stereochemistry and Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids

Cited by 60 publications

References 11 publications

Enzymatic Δ¹-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

Enzymatic Δ¹-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

The Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids. III. Kinetics and Isotope Effects^*

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

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Concerning the Stereochemistry and Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids

Cited by 60 publications

References 11 publications

Enzymatic Δ1-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

Enzymatic Δ1-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

The Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids. III. Kinetics and Isotope Effects*

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

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Product

Resources

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Enzymatic Δ¹-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

Enzymatic Δ¹-Dehydrogenation of 3-Ketosteroids—Reconciliation of Kinetic Isotope Effects with the Reaction Mechanism

The Mechanism of the Bacterial C-1,2 Dehydrogenation of Steroids. III. Kinetics and Isotope Effects^*