7P18

Structure, Mutagenesis, and QM:MM Modeling of 3-Ketosteroid Delta 1 -Dehydrogenase from Sterolibacterium denitrificans ─The Role of a New Putative Membrane-Associated Domain and Proton-Relay System in Catalysis.

(2023) Biochemistry 62: 808-823

• PubMed: 36625854 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1021/acs.biochem.2c00576
• Primary Citation of Related Structures:  
  7P18


• PubMed Abstract: 
  

  3-Ketosteroid Δ ¹ -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 Δ ¹ -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 Δ ⁴ -3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of Δ G of binding and predicted rate of dehydrogenation.

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Organizational Affiliation: 

Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239Kraków, Poland.