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

Michał Glanowski,Maciej Szaleniec,Vicent Moliner,Magdalena Procner,Przemysław Mielczarek,Tomasz Borowski,Patrycja Wójcik,Maria Oszajca,Andrzej J Bojarski,Dawid Lupa,Katarzyna Świderek

doi:10.1021/acscatal.1c01479

Abstract

Δ1-Dehydrogenation of 3-ketosteroids catalyzed by flavin adenine dinucleotide (FAD)-dependent 3-ketosteroid dehydrogenases (Δ1-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 Δ1-dehydrogenation and demonstrate that substrate binding, steroid dehydrogenation, and enzyme reoxidation proceed at comparable rates.

Highlights

Steroids are one of the most important groups of drugs on the market, finding their application in treating inflammation and diseases of immune function, such as allergies, asthma, autoimmune diseases, and several mineral metabolism disorders like hyponatremia, hyperkalemia, osteoporosis, and hypotension as well as in birth control.[1]
Our kinetic experiments confirmed that Δ1-dehydrogenation catalyzed by 3-Δ1-KSTD is proceeded according to the Ping-Pong bi−bi mechanism (Figure 4), as previously observed for transhydrogenation by Itagaki et al.[17] where Vmax is equal to kcatPing-Pong[Et]
Our analysis demonstrated that only the energy landscape of our mechanism can reconcile the experimental kinetic isotope effect (KIE) results obtained for substitutions at both C1 and C2 positions, which both lead to KIE values significantly greater than one but much lower than the calculated intrinsic KIE

Summary

Introduction

Steroids are one of the most important groups of drugs on the market, finding their application in treating inflammation and diseases of immune function, such as allergies, asthma, autoimmune diseases, and several mineral metabolism disorders like hyponatremia, hyperkalemia, osteoporosis, and hypotension as well as in birth control.[1]. Microbial fermentation very often provides steroid active pharmaceutical ingredients (APIs) that can be further functionalized by chemical or biocatalytic means. One of such biocatalytic processes that has been extensively studied during the last 50 years is Δ1dehydrogenation of steroids catalyzed by 56 kDa Δ1ketosteroid dehydrogenase (Δ1-KSTD). Δ1-KSTDs are essential in the production of several steroid drugs, such as betamethasone,[5,6] boldenone,[7,8] prednisone,[5,9] dexamethasone,[10,11] and steroid APIs [4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD), and 9α-hydroxy-4-androstene-3,17-dione].12−14 Due to their high importance in steroid metabolism, and for the pharmaceutical industry, the flavin adenine dinucleotide (FAD)-dependent Δ1KSTDs are one of the best-studied steroid-degrading

Methods

Results

Conclusion