Ketosteroid Isomerase

From Proteopedia
Jump to navigationJump to search

Ketosteroid Isomerase

Introduction

Template:STRUCTURE 1isk Ketosteroid isomerase (KSI, EC#5.3.3.1) is an enzyme that catalyzes the isomerization of 3-oxo-Δ5 ketosteroids to their hormonally active Δ4-conjugated isomers, as illustrated below.[1], [2]

 

This reaction is essential in the biosynthesis of steroids in mammals where KSI is a membrane-bound complex.[3] In bacteria, however, KSI exists as a soluble protein is involves in catabolism of steroids.[3] It was first isolated in and has been extensively studied in Commamonas tetosteroni (TI), a bacteria that is capable of growth with testosterone as its sole carbon source.[4] Structural and kinetic studies of this and its homolog from Pseudomonas putida with which it shares 34% sequence and near identical structural homology.[1],[3] It is one of the most efficient known enzymes with an essentially diffusion limited rate of catalysis.[2],[5] It is capable of increasing the catalytic rate by eleven orders of magnitude.[6] The high degree of efficiency is believed to be due to a preference for the transition state to move towards products rather than reactants although the exact mechanism of this preference is unclear.[1] Its high catalytic efficiency and unique active site geometry have made it fertile ground for examining the validity of the low barrier hydrogen bond hypothesis[7] and electrostatic preorganization.[8].

Structure

Ketosteroid isomerase exits as a 28 kDa homodimeric protein, in which the two dimers related to each other via hydrophobic and electrostatic interactions.[5] Each monomer consists of a curved beta-sheets and three alpha-helices. These secondary structures define a conical closed barrel geometry, with one open and one closed end, and create a deep pocket in which the active site resides.[3],[9] This unique geometry is shared by several other proteins (scytalone dehydratase, nuclear transport factor 2, and naphthalene 1,2-dioxygenase), however, these molecules do not share functional or sequence homology. It is speculated that this unique protein structure may enable better binding of hydrophobic substrates such as steroids.[3]


 
Topology diagram of KSI's structure Helices A, B, and C are in an antiparallel arrangement. The six β-strands form a mixed β-sheet.

Alpha-Helices

Each monomer of KSI contains three α-helices. Helix-A is contains residues Thr3 to (N-cap) to Ala20. Helix-B contains residues Asp22(N-cap) to Phe30. Helix-C contains residues Thr48 to Leu61. [5]

Alpha-Helix Capping Motifs

α-helix capping motifs are defined by specific patterns of hydrophobic interactions and hydrogen bonding that occur at both the initiation and termination of these secondary structural elements. [10] Capping motifs can also help support the formation of tertiary structural elements. KSI contains examples several of these motifs involved in both C-terminal and N-teminal capping, with two illustrations of C-terminal capping motifs as illustrated below (nomenclature adapted from that of Aurora and Rose [10]):

 
Examples of C-terminal helix capping motifs present in KSI. (A) α-L capping motif. (B) Proline capping motif.
Alpha-L C-terminal capping motif

An α-L C-terminal motif is located at the terminus of helix A (Ala17 to Leu23) and is defined by the following amino acid sequence ALNA-GDLD where Ala20 is in the C-cap position. The α-L motif is stabilized by a hydrogen bond between the backbone carbonyl of Ala17 and the backbone amide hydrogen of Gly21. Furthermore, a hydrophobic interactions between Leu18 and Leu23 adds stability to this structure. The motif is somewhat constrained by the presence of only one residue (Gly21) between helices A and B.

Proline C-terminal capping motif

The presence of a proline at position 62 "breaks" helix C's secondary structure. Leu59 and Val65 form a hydrophobic stable to support this secondary structural element.

Alpha-Helix Packing

Helix B is packed between Helices A and C in a nearly anti-parrallel configuration. Helices B and C cross the enzymatically active cavity forming the cavity's "front face."[5]

Beta-Sheet

Each monomer of KSI contains a six-strand mixed beta-sheet. Strand 1 of the β-sheet is composed of residues Glu43 to Gly47. Strand 2 is contains residues Ala34 to Asp38 and runs antiparallel to strand 1. Leu63 to Val74 make up strand 3 which runs antiparallel to strand 4 (Glu77 to Tyr88). Residues Arg91 to Phe104 and Lys108 to Gly124 form strands 5 and 6 respectively. Strand 5 runs antiparallel to strand 4, while strand 6 runs antiparallel to strand 5 and parallel to strand 1.[5] Each β-sheet contains two β-bulges: Thr68 to Glu70 and Phe116 to Asn120. Both the β-bulges participate in dimer-dimer interactions, potentially leading to their stabilization. The juxtaposition of the two β-bulges on each side of the sheet with a central proline residue creates a substantial kink in the β-sheet.[5]

Beta-Turns and Loops

β-strand 1 is connected to β-strand 2 via a four residue linkage (Pro39 to Ser42) with Pro39 being in a cis-conformation. Two examples of type-II β-turns can also be observed in KSI-between β-stands 3 & 4 (Ala75-Asn76→see figure) and β-strands 4 & 5 (Gln89-Gly90). A loop structure (Asn105-Val107) also connects β-strands 5 and 6.

 
Example of Type-II β-turn between β-strands 3 & 4. A type II β-turn is formed by residues Val74 (i), Ala75 (i+1), Asn76 (i+2), and Glu77 (i+3). The -carbons of Val74 and Glu77, Ci and Ci+1 respectively, are located 4.8 Å apart in agreement with an average separation of 4.6 Å in a type II turn. The dihedral angles defining the -turn are also is agreement with idealized values with appropriate deviations (30o with one angle allowed to deviate by as much as 45o)—i+1=-50.5o, i+1=100.6o, i+2=37.5o, and i+2=34.2o compared to average experimental values of i+1=-60o, i+1=120o, i+2=80o, and i+2=0o. Both i+2 and i+2 deviate by only slightly more than 30o from their idealized values. Also defining this type II -turn is the presence of a hydrogen bond between the backbone carbonyl of Val74 (i) and the backbone amide hydrogen of Glu77 (i+3).
  1. ↑ 1.0 1.1 1.2 Pollack RM. Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase. Bioorg Chem. 2004 Oct;32(5):341-53. PMID:15381400 doi:10.1016/j.bioorg.2004.06.005
  2. ↑ 2.0 2.1 TALALAY P, WANG VS. Enzymic isomerization of delta5-3-ketosteroids. Biochim Biophys Acta. 1955 Oct;18(2):300-1. PMID:13276386
  3. ↑ 3.0 3.1 3.2 3.3 3.4 Ha NC, Choi G, Choi KY, Oh BH. Structure and enzymology of Delta5-3-ketosteroid isomerase. Curr Opin Struct Biol. 2001 Dec;11(6):674-8. PMID:11751047
  4. ↑ Ha NC, Choi G, Choi KY, Oh BH. Structure and enzymology of Delta5-3-ketosteroid isomerase. Curr Opin Struct Biol. 2001 Dec;11(6):674-8. PMID:11751047
  5. ↑ 5.0 5.1 5.2 5.3 5.4 5.5 Wu ZR, Ebrahimian S, Zawrotny ME, Thornburg LD, Perez-Alvarado GC, Brothers P, Pollack RM, Summers MF. Solution structure of 3-oxo-delta5-steroid isomerase. Science. 1997 Apr 18;276(5311):415-8. PMID:9103200
  6. ↑ Murzin AG. How far divergent evolution goes in proteins. Curr Opin Struct Biol. 1998 Jun;8(3):380-7. PMID:9666335
  7. ↑ Cleland WW, Frey PA, Gerlt JA. The low barrier hydrogen bond in enzymatic catalysis. J Biol Chem. 1998 Oct 2;273(40):25529-32. PMID:9748211
  8. ↑ Kraut DA, Sigala PA, Pybus B, Liu CW, Ringe D, Petsko GA, Herschlag D. Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole. PLoS Biol. 2006 Apr;4(4):e99. Epub 2006 Mar 28. PMID:16602823 doi:10.1371/journal.pbio.0040099
  9. ↑ Cho HS, Choi G, Choi KY, Oh BH. Crystal structure and enzyme mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni. Biochemistry. 1998 Jun 9;37(23):8325-30. PMID:9622484 doi:10.1021/bi9801614
  10. ↑ 10.0 10.1 Aurora R, Rose GD. Helix capping. Protein Sci. 1998 Jan;7(1):21-38. PMID:9514257 doi:10.1002/pro.5560070103

Proteopedia Page Contributors and Editors (what is this?)

Laura M. Haynes, Michal Harel, Joel L. Sussman, Alexander Berchansky

Retrieved from "https://proteopedia.org/index.php?title=Ketosteroid_Isomerase&oldid=1241055"