8CEI

Succinyl-CoA Reductase from Clostridium kluyveri (SucD)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.267 
  • R-Value Work: 0.242 
  • R-Value Observed: 0.243 

Starting Model: experimental
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wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

Enhancing the Substrate Specificity of Clostridium Succinyl-CoA Reductase for Synthetic Biology and Biocatalysis.

Pfister, P.Diehl, C.Hammarlund, E.Carrillo, M.Erb, T.J.

(2023) Biochemistry 62: 1786-1793

  • DOI: https://doi.org/10.1021/acs.biochem.3c00102
  • Primary Citation of Related Structures:  
    8CEI, 8CEJ, 8CEK

  • PubMed Abstract: 

    Succinyl-CoA reductase (SucD) is an acylating aldehyde reductase that catalyzes the NADPH-dependent reduction of succinyl-CoA to succinic semialdehyde. The reaction sequence from succinate to crotonyl-CoA is of particular interest for several new-to-nature CO 2 -fixation pathways, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, in which SucD plays a key role. However, pathways like the CETCH cycle feature several CoA-ester intermediates, which could be potentially side substrates for this enzyme. Here, we show that the side reaction for most CETCH cycle metabolites is relatively small (<2%) with the exception of mesaconyl-C1-CoA (16%), which represents a competing substrate in this pathway. We addressed this promiscuity by solving the crystal structure of a SucD of Clostridium kluyveri in complex with NADP + and mesaconyl-C1-CoA. We further identified two residues (Lys70 and Ser243) that coordinate mesaconyl-C1-CoA at the active site. We targeted those residues with site-directed mutagenesis to improve succinyl-CoA over mesaconyl-C1-CoA reduction. The best resulting SucD variant, K70R, showed a strongly reduced side activity for mesaconyl-C1-CoA, but the substitution also reduced the specific activity for succinyl-CoA by a factor of 10. Transferring the same mutations into a SucD homologue from Clostridium difficile similarly decreases the side reaction of this enzyme for mesaconyl-C1-CoA from 12 to 2%, notably without changing the catalytic efficiency for succinyl-CoA. Overall, our structure-based engineering efforts provided a highly specific enzyme of interest for several applications in biocatalysis and synthetic biology.


  • Organizational Affiliation

    Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Str. 10, 35043 Marburg, Germany.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Succinate-semialdehyde dehydrogenase (acetylating)
A, B, C, D
453Clostridium kluyveriMutation(s): 0 
Gene Names: sucDCKL_3015
EC: 1.2.1.76
UniProt
Find proteins for P38947 (Clostridium kluyveri (strain ATCC 8527 / DSM 555 / NCIMB 10680))
Explore P38947 
Go to UniProtKB:  P38947
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP38947
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.267 
  • R-Value Work: 0.242 
  • R-Value Observed: 0.243 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 86.231α = 90
b = 89.339β = 104.64
c = 137.322γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
XDSdata reduction
SCALAdata scaling
PHENIXphasing

Structure Validation

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Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
Max Planck SocietyGermany--

Revision History  (Full details and data files)

  • Version 1.0: 2023-05-31
    Type: Initial release
  • Version 1.1: 2023-06-14
    Changes: Database references
  • Version 1.2: 2023-09-20
    Changes: Data collection, Refinement description