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.
3-Ketosteroid Δ1-dehydrogenases (KstD)
are important
microbial flavin enzymes that initiate the metabolism of steroid ring
A and find application in the synthesis of steroid drugs. We present
a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated
domain and extended proton-relay system. The experimental and theoretical
studies show that the steroid Δ1-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. The modeling confirms the origin of the enantioselectivity
of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be
the best substrate of AcmB in terms of ΔG of
binding and predicted rate of dehydrogenation.
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