4RLQ

Crystal structure of a benzoate coenzyme A ligase with o-Toluic acid


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.63 Å
  • R-Value Free: 0.193 
  • R-Value Work: 0.158 
  • R-Value Observed: 0.160 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity.

Thornburg, C.K.Wortas-Strom, S.Nosrati, M.Geiger, J.H.Walker, K.D.

(2015) Biochemistry 54: 6230-6242

  • DOI: https://doi.org/10.1021/acs.biochem.5b00899
  • Primary Citation of Related Structures:  
    4EAT, 4RLF, 4RLQ, 4RM2, 4RM3, 4RMN, 4ZJZ

  • PubMed Abstract: 

    A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants K(app)cat and K(app)M were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Benzoate-coenzyme A ligase
A, B
519Rhodopseudomonas palustrisMutation(s): 0 
Gene Names: badA
UniProt
Find proteins for Q93TK0 (Rhodopseudomonas palustris)
Explore Q93TK0 
Go to UniProtKB:  Q93TK0
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ93TK0
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
3SK
Query on 3SK

Download Ideal Coordinates CCD File 
C [auth A],
F [auth B]
2-methylbenzoic acid
C8 H8 O2
ZWLPBLYKEWSWPD-UHFFFAOYSA-N
GOL
Query on GOL

Download Ideal Coordinates CCD File 
D [auth A]
E [auth A]
G [auth B]
H [auth B]
I [auth B]
D [auth A],
E [auth A],
G [auth B],
H [auth B],
I [auth B],
J [auth B],
K [auth B],
L [auth B],
M [auth B]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.63 Å
  • R-Value Free: 0.193 
  • R-Value Work: 0.158 
  • R-Value Observed: 0.160 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 58.659α = 90
b = 94.808β = 104.92
c = 95.346γ = 90
Software Package:
Software NamePurpose
HKL-2000data collection
Molmodel building
REFMACrefinement
HKL-2000data reduction
HKL-2000data scaling
Molphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2015-09-30
    Type: Initial release
  • Version 1.1: 2015-10-07
    Changes: Database references, Structure summary
  • Version 1.2: 2015-11-11
    Changes: Database references
  • Version 1.3: 2024-02-28
    Changes: Data collection, Database references, Derived calculations