Structure, mutagenesis and QM:MM modelling of 3-ketosteroid Δ1-dehydrogenase from Sterolibacterium denitrificans – the role of new membrane-associated domain and proton-relay system in catalysis

Abstract

3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and are useful find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid 1,2-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium substituted substrates. The role of the active site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. Finally, the modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ1-3- ketosteroids.

1,2-Hydrogenation and Transhydrogenation Catalyzed by 3-Ketosteroid Δ1-Dehydrogenase from Sterolibacterium denitrificans—Kinetics, Isotope Labelling and QM:MM Modelling Studies

“…This issue, unfortunately, is far more complicated. Recently, we demonstrated with pre-steady-state kinetics of the half-reaction cycles (i.e., reductive (RHR) or oxidative (OHR) half-reaction cycles), that the pH dependency of dehydrogenation was flat between 7.6 and 10.0, with a small pH optimum at pH 9.0, while it was the oxidation of the reduced enzyme with DCPIP which proceeded preferentially under slightly acidic conditions [ 9 ]. Therefore, it seems that the Y363/proton relay system can act as a deprotonated base in a very wide pH range, and the sharp rise in the activity at pK a1 of 5.5 is associated with the DCPIP reduction/FADH − oxidation process.…”

“…An example of such a process is the dehydrogenation of 4-androsten-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) [ 3 , 4 ]. The dehydrogenation of AD and other 3-ketosteroids, such as 9α-hydroxy-4-androstene-3,17-dione [ 5 , 6 ], catalyzed by KstDs, is a critical reaction in the environmental cholesterol degradation pathway, initiating a ring A opening pathway and a central degradation pathway of the sterane ring system [ 7 , 8 , 9 ]. It also has industrial applications in the production of API and synthones for further modification [ 1 ].…”

“…Based on the structural studies supported by site-directed mutagenesis, kinetic isotope effect (KIE) studies and QM:MM MD modelling, the reaction mechanism of 1,2-dehydrogenation catalyzed by AcmB was described in detail. 1,2-Dehydrogenation catalyzed by KstDs proceeds according to ping-pong bi-bi kinetic mechanisms, which can be divided into oxidative half-reaction (OHR), during which the steroid is oxidized and the FAD is reduced, and reductive half-reaction (RHR), during which the other substrate (a natural or artificial electron acceptor such as, e.g., 2,6-dichloroindophenol) is reduced and the FADH − is reoxidized [ 9 ]. The RHR proceeds according to the previously suggested Ec2b two-step mechanism [ 1 , 9 , 14 ].…”

“…1,2-Dehydrogenation catalyzed by KstDs proceeds according to ping-pong bi-bi kinetic mechanisms, which can be divided into oxidative half-reaction (OHR), during which the steroid is oxidized and the FAD is reduced, and reductive half-reaction (RHR), during which the other substrate (a natural or artificial electron acceptor such as, e.g., 2,6-dichloroindophenol) is reduced and the FADH − is reoxidized [ 9 ]. The RHR proceeds according to the previously suggested Ec2b two-step mechanism [ 1 , 9 , 14 ]. The reaction starts when deprotonated tyrosine (Y363), acting as a base, enantioselectively abstracts 2β protons from the steroid and leads to the formation of the carbanion intermediate, stabilized by the H-bond with tyrosine residues present at the active site (Tyr536, Tyr118 and Y363).…”

“…In the next step, the hydride is transferred from the 1α position to N5 of the FAD. After the release of the 1-dehydro-steroid in RHR, the re-oxidant binds to the active site and reoxidizes FADH − to FAD [ 9 ].…”