5KCZ

horse liver S48T alcohol dehydrogenase complexed with NAD and trifluoroethanol

  • Classification: OXIDOREDUCTASE
  • Organism(s): Equus caballus
  • Expression System: Escherichia coli
  • Mutation(s): Yes 

  • Deposited: 2016-06-07 Released: 2016-06-29 
  • Deposition Author(s): Plapp, B.V.
  • Funding Organization(s): National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.14 Å
  • R-Value Free: 0.148 
  • R-Value Work: 0.126 
  • R-Value Observed: 0.126 

Starting Model: experimental
View more details

wwPDB Validation   3D Report Full Report


Ligand Structure Quality Assessment 


This is version 1.5 of the entry. See complete history


Literature

Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93.

Kim, K.Plapp, B.V.

(2017) Chem Biol Interact 276: 77-87

  • DOI: https://doi.org/10.1016/j.cbi.2016.12.016
  • Primary Citation of Related Structures:  
    5KCP, 5KCZ

  • PubMed Abstract: 

    The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/K m ) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD + and TFE or PFB are very similar to the structures for the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/K m ) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/K m for (S)-2-alcohols without changing V/K m for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme and that dynamics must affect the rates of catalysis.


  • Organizational Affiliation

    Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, USA. Electronic address: [email protected].


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Alcohol dehydrogenase E chain
A, B
374Equus caballusMutation(s): 1 
EC: 1.1.1.1
UniProt
Find proteins for P00327 (Equus caballus)
Explore P00327 
Go to UniProtKB:  P00327
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00327
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
NAJ
Query on NAJ

Download Ideal Coordinates CCD File 
E [auth A],
L [auth B]
NICOTINAMIDE-ADENINE-DINUCLEOTIDE (ACIDIC FORM)
C21 H27 N7 O14 P2
BAWFJGJZGIEFAR-NNYOXOHSSA-N
MRD
Query on MRD

Download Ideal Coordinates CCD File 
G [auth A],
H [auth A],
I [auth A],
N [auth B]
(4R)-2-METHYLPENTANE-2,4-DIOL
C6 H14 O2
SVTBMSDMJJWYQN-RXMQYKEDSA-N
ETF
Query on ETF

Download Ideal Coordinates CCD File 
F [auth A],
M [auth B]
TRIFLUOROETHANOL
C2 H3 F3 O
RHQDFWAXVIIEBN-UHFFFAOYSA-N
ZN
Query on ZN

Download Ideal Coordinates CCD File 
C [auth A],
D [auth A],
J [auth B],
K [auth B]
ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.14 Å
  • R-Value Free: 0.148 
  • R-Value Work: 0.126 
  • R-Value Observed: 0.126 
  • Space Group: P 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 44.4α = 91.97
b = 51.38β = 103.03
c = 92.29γ = 110.28
Software Package:
Software NamePurpose
REFMACrefinement
d*TREKdata reduction
d*TREKdata scaling
REFMACphasing

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data

  • Released Date: 2016-06-29 
  • Deposition Author(s): Plapp, B.V.

Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)United StatesGM078446

Revision History  (Full details and data files)

  • Version 1.0: 2016-06-29
    Type: Initial release
  • Version 1.1: 2017-07-05
    Changes: Database references, Derived calculations, Refinement description
  • Version 1.2: 2017-09-27
    Changes: Author supporting evidence
  • Version 1.3: 2017-11-22
    Changes: Database references
  • Version 1.4: 2019-12-25
    Changes: Author supporting evidence
  • Version 1.5: 2023-09-27
    Changes: Data collection, Database references, Derived calculations, Refinement description