Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ¹-dehydrogenase fromRhodococcus erythropolisSQ1

Abstract

3-Ketosteroid Á 1 -dehydrogenase plays a crucial role in the early steps of steroid degradation by introducing a double bond between the C1 and C2 atoms of the A-ring of its 3-ketosteroid substrates. The 3-ketosteroid Á 1 -dehydrogenase from Rhodococcus erythropolis SQ1, a 56 kDa flavoprotein, was crystallized using the sitting-drop vapour-diffusion method at room temperature. The crystals grew in various buffers over a wide pH range (from pH 5.5 to 10.5), but the best crystallization condition consisted of 2%(v/v) PEG 400, 0.1 M HEPES pH 7.5, 2.0 M ammonium sulfate. A native crystal diffracted X-rays to 2.0 Å resolution. It belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 107.4, b = 131.6, c = 363.2 Å , and contained eight molecules in the asymmetric unit. The initial structure of the enzyme was solved using multiwavelength anomalous dispersion (MAD) data collected from a Pt-derivatized crystal.

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

“…Protein Expression and Purification-A protocol for ⌬ 1 -KSTD1 expression has been established (2), as well as a procedure for its purification (17). In brief, Escherichia coli strain BL21(DE3) cells harboring the pET15b-kstD1 plasmid (2) were grown at 290 K. N-terminally His-tagged ⌬ 1 -KSTD1 was overexpressed by induction with 100 M isopropyl-␤-D-1-thiogalactopyranoside for 48 h. After cell lysis, ⌬ 1 -KSTD1 was purified by immobilized Ni 2ϩ affinity chromatography (HisTrap HP; GE Healthcare), followed by anion exchange chromatography (Resource Q; GE Healthcare) and size exclusion chromatography (Superdex 200 10/300 GL; GE Healthcare).…”

“…Protein Crystallization-⌬ 1 -KSTD1 was crystallized using the sitting drop, vapor diffusion method at room temperature (17). Typically, 0.30-l drops comprising 0.15 l of protein sample (15 mg ml Ϫ1 protein) mixed with 0.15 l of reservoir solution (2% (v/v) polyethylene glycol 400, 0.1 M HEPES were equilibrated against wells containing 50 l of the reservoir solution.…”

“…X-ray Data Collection and Processing-Before x-ray diffraction data collection, ⌬ 1 -KSTD1 crystals were transferred for 5 s to a cryoprotection solution (reservoir solution with 40% (w/v) sucrose added), followed by a 1-s transfer to a 1:1 solution of paraffin oil and paratone-N and flash cooling in liquid nitrogen (17). ⌬ 1 -KSTD1 crystals with bound ADD (⌬ 1 -KSTD1⅐ADD) were cryoprotected in a similar way, except that both above solutions were saturated overnight with ADD powder.…”

“…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.…”

“…Previously, we described the purification, crystallization, and preliminary x-ray crystallographic analysis of ⌬ 1 -KSTD1 (17). Here, we report crystal structures of the enzyme in the unliganded form and in complex with the reaction product 1,4-androstadiene-3,17-dione (ADD).…”