6MSW

Crystal structure of BH1352 2-deoxyribose-5-phosphate from Bacillus halodurans, K184L mutant


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.17 Å
  • R-Value Free: 0.211 
  • R-Value Work: 0.193 
  • R-Value Observed: 0.193 

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This is version 1.3 of the entry. See complete history


Literature

Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol.

Kim, T.Stogios, P.J.Khusnutdinova, A.N.Nemr, K.Skarina, T.Flick, R.Joo, J.C.Mahadevan, R.Savchenko, A.Yakunin, A.F.

(2020) J Biol Chem 295: 597-609

  • DOI: https://doi.org/10.1074/jbc.RA119.011363
  • Primary Citation of Related Structures:  
    6D33, 6MSW

  • PubMed Abstract: 

    Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of ( R )-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans , TM1559 from Thermotoga maritima , and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr 224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys 155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.


  • Organizational Affiliation

    Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada; Future Technology Center, LG Chem, Gangseo-gu, Seoul 150-721, Korea.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Deoxyribose-phosphate aldolase
A, B, C, D, E
A, B, C, D, E, F
224Halalkalibacterium halodurans C-125Mutation(s): 1 
Gene Names: deoCdraBH1352
EC: 4.1.2.4
UniProt
Find proteins for Q9KD67 (Halalkalibacterium halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125))
Explore Q9KD67 
Go to UniProtKB:  Q9KD67
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9KD67
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
GOL
Query on GOL

Download Ideal Coordinates CCD File 
G [auth A]
H [auth A]
I [auth A]
J [auth B]
K [auth B]
G [auth A],
H [auth A],
I [auth A],
J [auth B],
K [auth B],
L [auth B],
M [auth C],
N [auth C],
O [auth C],
P [auth D],
Q [auth D],
R [auth D],
S [auth D],
T [auth E],
U [auth E],
V [auth F],
W [auth F]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.17 Å
  • R-Value Free: 0.211 
  • R-Value Work: 0.193 
  • R-Value Observed: 0.193 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 240.779α = 90
b = 55.125β = 127.71
c = 176.297γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
PHENIXmodel building
Cootmodel building

Structure Validation

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Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
Natural Sciences and Engineering Research Council (NSERC, Canada)CanadaIndustrial Biocatalysis Network

Revision History  (Full details and data files)

  • Version 1.0: 2019-10-23
    Type: Initial release
  • Version 1.1: 2020-01-08
    Changes: Author supporting evidence
  • Version 1.2: 2020-05-06
    Changes: Database references
  • Version 1.3: 2023-10-11
    Changes: Data collection, Database references, Refinement description