3UZY

Crystal structure of 5beta-reductase (AKR1D1) E120H mutant in complex with NADP+ and 5beta-dihydrotestosterone


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.83 Å
  • R-Value Free: 0.218 
  • R-Value Work: 0.173 
  • R-Value Observed: 0.175 

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Ligand Structure Quality Assessment 


This is version 1.3 of the entry. See complete history


Literature

Conversion of Human Steroid 5beta-Reductase (AKR1D1) into 3β-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: EXAMPLE OF PERFECT ENZYME ENGINEERING.

Chen, M.Drury, J.E.Christianson, D.W.Penning, T.M.

(2012) J Biol Chem 287: 16609-16622

  • DOI: https://doi.org/10.1074/jbc.M111.338780
  • Primary Citation of Related Structures:  
    3UZW, 3UZX, 3UZY, 3UZZ

  • PubMed Abstract: 

    Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ(4)-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.


  • Organizational Affiliation

    Department of Pharmacology and Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
3-oxo-5-beta-steroid 4-dehydrogenase
A, B
346Homo sapiensMutation(s): 1 
Gene Names: AKR1D1SRD5B1
EC: 1.3.1.3
UniProt & NIH Common Fund Data Resources
Find proteins for P51857 (Homo sapiens)
Explore P51857 
Go to UniProtKB:  P51857
PHAROS:  P51857
GTEx:  ENSG00000122787 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP51857
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.83 Å
  • R-Value Free: 0.218 
  • R-Value Work: 0.173 
  • R-Value Observed: 0.175 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 50.015α = 90
b = 110.224β = 90
c = 129.497γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
PHENIXrefinement
PDB_EXTRACTdata extraction
HKL-2000data collection
HKL-2000data reduction
CNSphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2012-03-21
    Type: Initial release
  • Version 1.1: 2012-06-27
    Changes: Database references
  • Version 1.2: 2017-11-08
    Changes: Refinement description
  • Version 1.3: 2023-09-13
    Changes: Data collection, Database references, Derived calculations, Refinement description