7P18

Crystal structure of 3-ketosteroid delta1-dehydrogenase from Sterolibacterium denitrificans in complex with 1,4-androstadiene-3,17-dione

7P18 の概要

• エントリーDOI: 10.2210/pdb7p18/pdb
• 分子名称: 3-oxosteroid 1-dehydrogenase, FLAVIN-ADENINE DINUCLEOTIDE, ANDROSTA-1,4-DIENE-3,17-DIONE, ... (7 entities in total)
• 機能のキーワード: flavoprotein, dehydrogenase, oxidoreductase, 3-ketosteroid delta1-dehydrogenase, 3-ketosteroid dehydrogenase, 3-oxosteroid dehydrogenase, rossmann fold
• 由来する生物種: Sterolibacterium denitrificans
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 125541.08

構造登録者

Wojcik, P.,Mrugala, B.,Kurpiewska, K.,Szaleniec, M. (登録日: 2021-07-01, 公開日: 2021-07-21, 最終更新日: 2024-02-07)

主引用文献

Wojcik, P.,Glanowski, M.,Mrugala, B.,Procner, M.,Zastawny, O.,Flejszar, M.,Kurpiewska, K.,Niedzialkowska, E.,Minor, W.,Oszajca, M.,Bojarski, A.J.,Wojtkiewicz, A.M.,Szaleniec, M.
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.
Biochemistry, 62:808-823, 2023

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

PubMed: 36625854
DOI: 10.1021/acs.biochem.2c00576

実験手法

X-RAY DIFFRACTION (1.84 Å)