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

12 December 2022, Version 2

Abstract

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 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 modelling 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 modelling 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.

Keywords

ketosteroid Δ1-dehydrogenase
3-ketosteroids
Δ1-dehydrogenation
1(2)-dehydrogenation
kinetic isotope effect
3-ketosteroid dehydrogenase

Supplementary materials

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Description
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Supplementary Information
Description
Extended experimental procedure (Site-directed mutagenesis, Kinetic isotope effect– competition method, QMMM model setup), phylogenetic tree for ‘loop’ sequences of KstDs and six-tyrosine motif analysis, sequence alignment of 82 KstDs, diffraction data collection and refinement statistics, Size-exclusion chromatograms of AcmB, figures and mobility analyses of putative membrane-associated domain, figures of AcmB structure, binding sites, details on MMPBSA DGbinding and interaction energies for AcmB and KstD1, details on MD trajectories, figures of stationary states of all mechanisms and figures depicting PES profiles, detailed data on activities of mutants, experimental and theoretical kinetic isotope effects, PDB files of representative structures optimized at the B3LYP/AMBER level of theory
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SI - population analysis
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Population analysis
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