Steroid Delta-isomerase

steroid delta-isomerase

Crystallographic structure of Pseudomonas putida steroid Δ5-isomerase homodimer.[1]
Identifiers
EC number 5.3.3.1
CAS number 9031-36-1
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO

In enzymology, a steroid Δ5-isomerase (EC 5.3.3.1) is an enzyme that catalyzes the chemical reaction

a 3-oxo-Δ5-steroid a 3-oxo-Δ4-steroid

Hence, this enzyme has one substrate, a 3-oxo-Δ5-steroid, and one product, a 3-oxo-Δ4-steroid.

Introduction

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).[2] 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.[3] Mammalian KSI has been studied from bovine adrenal cortex[4] and rat liver.[5] 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.[6] The above reaction in the absence of enzyme takes 7 weeks to complete in aqueous solution.[7] KSI performs this reaction on an order of 1011 times faster, ranking it among the most proficient enzymes known.[7] Bacterial KSI also serves as a model protein for studying enzyme catalysis[8] and protein folding.[9]

Structural studies

KSI exists as a homodimer with two identical halves.[9] 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.[9] Results show that the dimerization is essential to function.[9] 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.[10]

No complete atomic structure of KSI appeared until 1997, when an NMR structure of TI KSI was reported.[11] 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.[11] 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.

Mechanism

A schematic description of the isomerization catalyzed by C. testosteroni steroid delta-isomerase.

KSI catalyzes a C-H bond cleavage and formation through an enolate intermediate at a diffusion-limited rate.[2] 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.[2] Tyr-14 and Asp-99 are positioned deep within the hydro-phobic active site and form a so-called oxanion hole.[12] Protonated Asp-38 then transfers its proton to position 6 of the steroid ring to complete the reaction.[2] The hydrogen bonds from Tyr-14 and Asp-99 are known to significantly affect the rate of catalysis in KSI.[2]

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,[11][13] implicating Asp-99 as important for enzymatic activity. Wu et al.[11] 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.,[14] 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).[15] 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.[15][16] 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.[17]

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.[16][18]

There have been conflicting results on the ionization state of the intermediate, whether it exists as the enolate[19] or enol.[20] Pollack uses a thermodynamic argument to suggest the intermediate exists as the enolate.[2]

Biological Function

KSI occurs in animal tissues concerned with steroid hormone biosynthesis, such as the adrenal, testis, and ovary.[21] 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.[22] 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,[23] while in C. testosteroni and P. putida, Δ5,3-ketosteroid isomerase just transfers a double bond at Δ5 of 3-ketosteroid to Δ4.[24]

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.[25]

Model Enzyme

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.[10][12] 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.[26][27] 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.[28][29] 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.[8]

KSI has also been a model system for studying protein folding. Kim et al. studied the effect of folding and tertiary structure on the function of KSI.[9]

References

  1. ↑ PDB: 3VSY​; Kobe A, Caaveiro JM, Tashiro S, Kajihara D, Kikkawa M, Mitani T, Tsumoto K (February 2013). "Incorporation of Rapid Thermodynamic Data in Fragment-Based Drug Discovery". J Med Chem. 56 (5): 2155–9. doi:10.1021/jm301603n. PMID 23419007.
  2. 1 2 3 4 5 6 Pollack, R (2004). "Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase". Bioorganic Chemistry. 32 (5): 341–53. doi:10.1016/j.bioorg.2004.06.005. PMID 15381400.
  3. ↑
    Cho HS, Choi G, Choi KY, Oh BH (May 1998). "Crystal Structure and Enzyme Mechanism of Δ5-3-Ketosteroid Isomerase from Pseudomonas testosteroni". Biochemistry. 37 (23): 8325–30. doi:10.1021/bi9801614. PMID 9622484.
  4. ↑ Bertolino, A; Benson, A; Talalay, P (1979). "Activation of Δ5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins". Biochemical and Biophysical Research Communications. 88 (3): 1158–66. doi:10.1016/0006-291X(79)91530-4. PMID 465075.
  5. ↑ Benson, A; Talalay, P (1976). "Role of reduced glutathione in the Δ5-3-ketosteroid isomerase reaction of liver". Biochemical and Biophysical Research Communications. 69 (4): 1073–9. doi:10.1016/0006-291X(76)90482-4. PMID 6023.
  6. ↑ Talalay, Paul; Benson, Ann M (1972). "Δ5-3-Ketosteroid Isomerase". In Boyer, Paul D. The Enzymes. 6 (3rd ed.). Academic Press. pp. 591–618. ISBN 978-0-12-122706-7.
  7. 1 2 Radzicka, A; Wolfenden, R (1995). "A proficient enzyme". Science. 267 (5194): 90–3. doi:10.1126/science.7809611. PMID 7809611.
  8. 1 2 Kraut, Daniel A.; Sigala, Paul A.; Pybus, Brandon; Liu, Corey W.; Ringe, Dagmar; Petsko, Gregory A.; Herschlag, Daniel (2006). "Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole". PLoS Biology. 4 (4): e99. doi:10.1371/journal.pbio.0040099. PMC 1413570Freely accessible. PMID 16602823.
  9. 1 2 3 4 5 Kim, D.-H.; Nam, GH; Jang, DS; Yun, S; Choi, G; Lee, HC; Choi, KY (2001). "Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B". Protein Science. 10 (4): 741–52. doi:10.1110/ps.18501. PMC 2373975Freely accessible. PMID 11274465.
  10. 1 2 Ha, N.-C.; Kim, MS; Lee, W; Choi, KY; Oh, BH (2000). "Detection of Large pKa Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes". Journal of Biological Chemistry. 275 (52): 41100–6. doi:10.1074/jbc.M007561200. PMID 11007792.
  11. 1 2 3 4 Wu, Z. R.; Ebrahimian, S; Zawrotny, ME; Thornburg, LD; Perez-Alvarado, GC; Brothers, P; Pollack, RM; Summers, MF (1997). "Solution Structure of 3-Oxo-5-Steroid Isomerase". Science. 276 (5311): 415–8. doi:10.1126/science.276.5311.415. PMID 9103200.
  12. 1 2 Childs W, Boxer SG (February 2010). "Proton affinity of the oxyanion hole in the active site of ketosteroid isomerase.". Biochemistry. 49 (12): 2725–31. doi:10.1021/bi100074s. PMID 20143849.
  13. ↑
    Lora D. Thornburg; Frédéric Hénot; Diarmad P. Bash; David C. Hawkinson; Shawn D. Bartel; Ralph M. Pollack (1998). "Electrophilic Assistance by Asp-99 of 3-Oxo-Δ5-steroid Isomerase". Biochemistry. 37: 10499–10506. doi:10.1021/bi980099a. PMID 9671521.
  14. ↑ Zhao, Qinjian; Abeygunawardana, Chitrananda; Gittis, Apostolos G.; Mildvan, Albert S. (1997). "Hydrogen Bonding at the Active Site of Δ5-3-Ketosteroid Isomerase†". Biochemistry. 36 (48): 14616–26. doi:10.1021/bi971549m. PMID 9398180.
  15. 1 2
    Kim SW, Cha SS, Cho HS, Kim JS, Ha NC, Cho MJ, Joo S, Kim KK, Choi KY, Oh BH (November 1997). "High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue.". Biochemistry. 36 (46): 14030–6. doi:10.1021/bi971546+. PMID 9369474.
  16. 1 2
    Zhao Q, Li YK, Mildvan AS, Talalay P (May 1995). "Ultraviolet spectroscopic evidence for decreased motion of the active site tyrosine residue of delta 5-3-ketosteroid isomerase by steroid binding.". Biochemistry. 34 (19): 6562–72. doi:10.1021/bi00019a038. PMID 7756287.
  17. ↑
    Sigala PA, Fafarman AT, Bogard PE, Boxer SG, Herschlag D (October 2007). "Do ligand binding and solvent exclusion alter the electrostatic character within the oxyanion hole of an enzymatic active site?". J Am Chem Soc. 129 (40): 12104–5. doi:10.1021/ja075605a. PMID 17854190.
  18. ↑
    Zhao Q, Abeygunawardana C, Mildvan AS (February 1996). "13C NMR relaxation studies of backbone and side chain motion of the catalytic tyrosine residue in free and steroid-bound delta 5-3-ketosteroid isomerase.". Biochemistry. 35 (5): 1525–32. doi:10.1021/bi9525381. PMID 8634283.
  19. ↑ Xue, Liang; Kuliopulos, Athan; Mildvan, Albert S.; Talalay, Paul (1991). "Catalytic mechanism of an active-site mutant (D38N) of .DELTA.5-3-ketosteroid isomerase". Biochemistry. 30 (20): 4991–7. doi:10.1021/bi00234a022. PMID 2036366.
  20. ↑ Petrounia, Ioanna P.; Pollack, Ralph M. (1998). "Substituent Effects on the Binding of Phenols to the D38N Mutant of 3-Oxo-Δ5-steroid Isomerase. A Probe for the Nature of Hydrogen Bonding to the Intermediate". Biochemistry. 37 (2): 700–5. doi:10.1021/bi972262s. PMID 9425094.
  21. ↑ Kawahara, Frank S.; Wang, Shu-Fang; Talalay, Paul (1962). "The Preparation and Properties of Crystalline Δ5-3-Ketosteroid Isomerase". The Journal of Biological Chemistry. 237: 1500–6. PMID 14454546.
  22. ↑ Talalay, Paul; Dobson, Marie Mollomo; Tapley, Donald F. (1952). "Oxidative Degradation of Testosterone by Adaptive Enzymes". Nature. 170 (4328): 620–1. doi:10.1038/170620a0. PMID 13002385.
  23. ↑
    Lachance Y, Luu-The V, Labrie C, Simard J, Dumont M, de Launoit Y, Guérin S, Leblanc G, Labrie F (February 1992). "Characterization of human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase gene and its expression in mammalian cells.". J Biol Chem. 267 (5): 3551. PMID 1737804.
  24. ↑ Horinouchi M, Hayashi T, Kudo T (2012). "Steroid degradation in Comamonas testosteroni.". J Steroid Biochem Mol Biol. 129 (1-2): 4–14. doi:10.1016/j.jsbmb.2010.10.008. PMID 21056662.
  25. ↑ Horinouchi M, Kurita T, Hayashi T, Kudo T (2010). "Steroid degradation genes in Comamonas testosteroni TA441: Isolation of genes encoding a Δ4(5)-isomerase and 3α- and 3β-dehydrogenases and evidence for a 100 kb steroid degradation gene hot spot.". J Steroid Biochem Mol Biol. 122 (4): 253–63. doi:10.1016/j.jsbmb.2010.06.002. PMID 20554032.
  26. ↑ Gerlt JA, Gassman PG (November 1993). "Understanding the rates of certain enzyme-catalyzed reactions: proton abstraction from carbon acids, acyl-transfer reactions, and displacement reactions of phosphodiesters.". Biochemistry. 32 (45): 11943–52. doi:10.1021/bi00096a001. PMID 8218268.
  27. ↑ Gerlt JA, Gassman PG (December 1993). "An explanation for rapid enzyme-catalyzed proton abstraction from carbon acids: importance of late transition states in concerted mechanisms.". Biochemistry. doi:10.1021/ja00077a062.
  28. ↑ Warshel A, Papazyan A, Kollman PA (July 1995). "On low-barrier hydrogen bonds and enzyme catalysis.". Science. 269 (5220): 102–6. doi:10.1126/science.7661987. PMID 7661987.
  29. ↑ Guthrie JP (March 1996). "Short strong hydrogen bonds: can they explain enzymic catalysis?". Chem Biol. 3 (3): 163–70. doi:10.1016/s1074-5521(96)90258-6. PMID 8807842.

Further reading

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