Crystallographic structure of Pseudomonas putida steroid Δ5-isomerase homodimer.
|PDB structures||RCSB PDB PDBe PDBsum|
|Gene Ontology||AmiGO / EGO|
- a 3-oxo-Δ5-steroid a 3-oxo-Δ4-steroid
This enzyme belongs to the family of isomerases, specifically those intramolecular oxidoreductases transposing C=C bonds. The systematic name of this enzyme class is 3-oxosteroid Δ5-Δ4-isomerase. Other names in common use include ketosteroid isomerase (KSI), hydroxysteroid isomerase, steroid isomerase, Δ5-ketosteroid isomerase, Δ5(or Δ4)-3-keto steroid isomerase, Δ5-steroid isomerase, 3-oxosteroid isomerase, Δ5-3-keto steroid isomerase, and Δ5-3-oxosteroid isomerase.
KSI has been studied extensively from the bacteria Comamonas testosteroni (TI), formerly referred to as Pseudomonas testosteroni, and Pseudomonas putida (PI). The enzymes from these two sources are 34% homologous, and structural studies have shown that the placement of the catalytic groups in the active sites is virtually identical. Mammalian KSI has been studied from bovine adrenal cortex and rat liver. This enzyme participates in c21-steroid hormone metabolism and androgen and estrogen metabolism. An example substrate is Δ5-androstene-3,17-dione, which KSI converts to Δ4-androstene-3,17-dione. The above reaction in the absence of enzyme takes 7 weeks to complete in aqueous solution. KSI performs this reaction on an order of 1011 times faster, ranking it among the most proficient enzymes known. Bacterial KSI also serves as a model protein for studying enzyme catalysis and protein folding.
KSI exists as a homodimer with two identical halves. The interface between the two monomers is narrow and well defined, consisting of neutral or apolar amino acids, suggesting the hydrophobic interaction is important for dimerization. Results show that the dimerization is essential to function. The active site is highly apolar and folds around the substrate in a manner similar to other enzymes with hydrophobic substrates, suggesting this fold is characteristic for binding hydrophobic substrates.
No complete atomic structure of KSI appeared until 1997, when an NMR structure of TI KSI was reported. This structure showed that the active site is a deep hydrophobic pit with Asp-38 and Tyr-14 located at the bottom of this pit. The structure is thus entirely consistent with the proposed mechanistic roles of Asp-38 and Tyr-14.
As of late 2007, 25 structures have been solved for this class of enzymes, with PDB accession codes 1BUQ, 1C7H, 1CQS, 1DMM, 1DMN, 1DMQ, 1E97, 1GS3, 1ISK, 1K41, 1OCV, 1OGX, 1OGZ, 1OH0, 1OHO, 1OHP, 1OHS, 1OPY, 1VZZ, 1W00, 1W01, 1W02, 1W6Y, 2PZV, and 8CHO.
KSI catalyzes a C-H bond cleavage and formation through an enolate intermediate at a diffusion-limited rate. The general base Asp-38 abstracts a proton from position 4 of the steroid ring to form an enolate that is stabilized by the hydrogen bond donating Tyr-14 and Asp-99. Tyr-14 and Asp-99 are positioned deep within the hydro-phobic active site and form a so-called oxanion hole. Protonated Asp-38 then transfers its proton to position 6 of the steroid ring to complete the reaction. The hydrogen bonds from Tyr-14 and Asp-99 are known to significantly affect the rate of catalysis in KSI.
The active site pit is lined with hydrophobic residues, but there exists an ionic residue, Asp-99, located adjacent to Tyr-14 and within hydrogen bonding distance of O-3. Mutagenesis of this residue to alanine (D99A) or asparagine (D99N) results in a loss in activity at pH 7 of 3000-fold and 27-fold, respectively, implicating Asp-99 as important for enzymatic activity. Wu et al. proposed a mechanism that involves both Tyr-14 and Asp-99 forming hydrogen bonds directly to O-3 of the steroid. This mechanism was challenged by Zhao et al., who postulated a hydrogen bonding network with Asp-99 hydrogen bonding to Tyr-14, which in turn forms a hydrogen bond to O-3.
Numerous physical changes occur upon steroid binding within the KSI active site. In the free enzyme an ordered water molecule is positioned within hydrogen-bonding distance of Tyr-16 (the PI equivalent of TI KSI Tyr-14) and Asp-103 (the PI equivalent of TI KSI Asp-99). This and additional disordered water molecules present within the unliganded active site are displaced upon steroid binding and are substantially excluded by the dense constellation of hydrophobic residues that pack around the bound, hydrophobic steroid skeleton. Sigala et al. found that solvent exclusion and replacement by the remote hydrophobic steroid rings negligibly alter the electrostatic environment within the KSI oxyanion hole.
Ligand binding does not grossly alter the conformations of backbone and side chain groups observed in X-ray structures of PI KSI. However, NMR and UV studies suggest that steroid binding restricts the motions of several active-site groups, including Tyr-16.
There have been conflicting results on the ionization state of the intermediate, whether it exists as the enolate or enol. Pollack uses a thermodynamic argument to suggest the intermediate exists as the enolate.
KSI occurs in animal tissues concerned with steroid hormone biosynthesis, such as the adrenal, testis, and ovary. KSI in Comamomas testosteroni is used in the degradation pathway of steroids, allowing this bacteria to utilize steroids containing a double bond at Δ5, such as testosterone, as its sole source of carbon. In mammals, transfer of a double bond at Δ5 to Δ4 is catalyzed by 3-β-hydroxy-Δ5-steroid dehydrogenase at the same time as the dehydroxylation of 3-β-hydroxyl group to ketone group, while in C. testosteroni and P. putida, Δ5,3-ketosteroid isomerase just transfers a double bond at Δ5 of 3-ketosteroid to Δ4.
A Δ5-3-ketosteroid isomerase-disrupted mutant of strain TA441 can grow on dehydroepiandrosterone, which has a double bond at Δ5, but cannot grow on epiandrosterone, which lacks a double bond at Δ5, indicating that C. testosteroni KSI is responsible for transfer of the double bond from Δ5 to Δ4 and transfer of the double bond by hydrogenation at Δ5 and following dehydrogenation at Δ4 is not possible.
KSI has been used as a model system to test different theories to explain how enzymes achieve their catalytic efficiency. Low-barrier hydrogen bonds and unusual pKa values for the catalytic residues have been proposed as the basis for the fast action of KSI. Gerlt and Gassman proposed the formation of unusually short, strong hydrogen bonds between KSI oxanion hole and the reaction intermediate as a means of catalytic rate enhancement. In their model, high-energy states along the reaction coordinate are specifically stabilized by the formation of these bonds. Since then, the catalytic role of short, strong hydrogen bonds has been debated. Another proposal explaining enzyme catalysis tested through KSI is the geometrical complementarity of the active site to the transition state, which proposes the active site electrostatics is complementary to the substrate transition state.
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- Steroid Isomerases at the US National Library of Medicine Medical Subject Headings (MeSH)