Abstract
3-Ketosteroid Δ(1)-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ(1)-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ(1)-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr(119), Tyr(318), Tyr(487), and Gly(491). Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr(318), respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr(487) and the backbone amide of Gly(491). Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr(487) and Gly(491) work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr(119), the general base Tyr(318) abstracts the axial β-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion.
Highlights
The values in parenthesis are for the highest resolution shell
Ii(h) is the integrated intensity of a reflection, ϽI(h)Ͼ is the mean intensity of multiple corresponding symmetry-related reflections, and N is the multiplicity of the given reflections
The crystal structure of ⌬1-KSTD1 was solved using multiwavelength anomalous dispersion data collected from a Pt-derivatized crystal [17], whereas that of ⌬1-KSTD11⁄7ADD was elucidated by rigid body fitting using the refined model of the holoenzyme and subsequent refinement
Summary
Protein Expression and Purification—A protocol for ⌬1-KSTD1 expression has been established [2], as well as a procedure for its purification [17]. Structure Determination and Refinement—The initial phases of ⌬1-KSTD1 were obtained using multiwavelength anomalous dispersion data collected from a Pt-derivatized crystal, and a complete model for the protein was obtained [17]. This model was subjected to rigid body refinement with the program Refmac5 [25] from the CCP4 package using native diffraction data of ⌬1-KSTD1. The plasmid template was digested using DpnI, and each of the mutants was transformed into E. coli XL1-Blue supercompetent cells (Stratagene) Individual clones of these E. coli variants were grown, and their plasmids were isolated for sequencing and transformation into the expression strain E. coli BL21(DE3). The kinetics parameters were analyzed by nonlinear regression curve fitting of the data to the MichaelisMenten equation using GraphPad Prism version 5.00 for Windows (GraphPad Software)
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