Delta 5-3-ketosteroid isomerase

 

Delta5-3 ketosteroid isomerase catalyses the isomerisation of delta5-3-ketosteroid to delta4-3-ketosteroid by intramolecular transfer of the C4beta proton to the C6beta position via a dienolic intermediate. The mechanism proceeds at a diffusion controlled limit within an environment shielded from solvent.

In mammals, this reaction is an essential step in the biosynthesis of steroid hormones and is catalysed by a membrane-bound complex. In bacteria, the enzyme appears to play a metabolic role in the degrading steroid substrates.

 

Reference Protein and Structure

Sequence
P07445 UniProt (5.3.3.1) IPR009959 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas putida (Bacteria) Uniprot
PDB
1e3v - Crystal structure of ketosteroid isomerase from Psedomonas putida complexed with deoxycholate (2.0 Å) PDBe PDBsum 1e3v
Catalytic CATH Domains
3.10.450.50 CATHdb (see all for 1e3v)

Enzyme Reaction (EC:5.3.3.1)

3-oxo-Delta(5)-steroid
CHEBI:47907ChEBI
→
3-oxo-Delta(4) steroid
CHEBI:47909ChEBI
Alternative enzyme names: 3-oxosteroid isomerase, Delta(5)(or Delta(4))-3-keto steroid isomerase, Delta(5)-3-keto steroid isomerase, Delta(5)-3-oxosteroid isomerase, Delta(5)-ketosteroid isomerase, Delta(5)-steroid isomerase, Hydroxysteroid isomerase, Steroid isomerase, Delta(5)-3-ketosteroid isomerase,

Enzyme Mechanism

Introduction

Asp40 initiates the reaction by abstracting the C4beta proton of the steroid substrate, deprotonating the C3-OH group. The activation energy of proton transfer is reduced by the presence of Asp103 and Tyr16 which form strong low barrier hydrogen bonds to the dienolyic intermediate, interactions which are stronger than the hydrogen bonding between to the initial substrate. The resulting dienolate intermediate is stabilised by 2 hydrogen bonds provided by Tyr16 and Asp103 to the C3 oxyanion. The proton is then relayed to C6, via conjugate attack of the enolate at the catalytic residue and the subsequent reketonisation results in protonation at the C6 position by Asp40.

Catalytic Residues Roles

UniProt PDB* (1e3v)
Asp40 Asp40A Deprotonates C4 to initiate the formation of the dienolate intermediate. It protonates C6 in reketonisation to yield the product. proton acceptor, proton donor
Tyr16 Tyr16A Stabilises the oxyanion transition state by forming a hydrogen bond to the C3 oxygen atom. Also acts as a general acid/base. proton acceptor, proton donor
Asp100 Asp100A Acts to stabilise the dienolic intermediate by forming low barrier hydrogen bonds. These interactions are also implicated in modifying the proton affinity of the substrate, allowing Asp 40 to act as a base at the alpha hydrogen to the carbonyl. electrostatic stabiliser
Asp103 Asp103A Stabilises the transition state. It has been shown to hydrogen bond directly to the steroid substrate, rather than indirectly via Tyr16 [PMID:10653633]. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, assisted keto-enol tautomerisation, overall reactant used, intermediate formation, overall product formed, native state of enzyme regenerated

References

  1. Ha NC et al. (2000), J Biol Chem, 275, 41100-41106. Detection of Large pK a Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes. DOI:10.1074/jbc.m007561200. PMID:11007792.
  2. Natarajan A et al. (2014), J Am Chem Soc, 136, 7643-7654. Using unnatural amino acids to probe the energetics of oxyanion hole hydrogen bonds in the ketosteroid isomerase active site. DOI:10.1021/ja413174b. PMID:24787954.
  3. van der Kamp MW et al. (2013), FEBS J, 280, 3120-3131. QM/MM modelling of ketosteroid isomerase reactivity indicates that active site closure is integral to catalysis. DOI:10.1111/febs.12158. PMID:23356661.
  4. Jang DS et al. (2004), Biochem J, 382, 967-973. Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase fromPseudomonas putidabiotype B. DOI:10.1042/bj20031871. PMID:15228388.
  5. Park H et al. (2003), J Am Chem Soc, 125, 901-911. Molecular dynamics and quantum chemical studies on the catalytic mechanism of Delta5-3-ketosteroid isomerase: the catalytic diad versus the cooperative hydrogen bond mechanism. DOI:10.1021/ja0208097. PMID:12537487.
  6. Choi G et al. (2000), Biochemistry, 39, 903-909. Asp-99 Donates a Hydrogen Bond Not to Tyr-14 but to the Steroid Directly in the Catalytic Mechanism of Δ5-3-Ketosteroid Isomerase fromPseudomonas putidaBiotype B†. DOI:10.1021/bi991579k. PMID:10653633.
  7. Kim SW et al. (1997), Biochemistry, 36, 14030-14036. High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue. DOI:10.1021/bi971546+. PMID:9369474.
  8. Wu ZR et al. (1997), Science, 276, 415-418. Solution Structure of 3-Oxo-5-Steroid Isomerase. DOI:10.1126/science.276.5311.415. PMID:9103200.
  9. Kuliopulos A et al. (1989), Biochemistry, 28, 149-159. Kinetic and ultraviolet spectroscopic studies of active-site mutants of .DELTA.5-3-ketosteroid isomerase. DOI:10.1021/bi00427a022. PMID:2706241.

Contributors

Sophie T. Williams, Anna Waters, Craig Porter, Mei Leung, Gemma L. Holliday, James Willey, Trung Nguyen