5KBZ

Structure of the PksA Product Template domain in complex with a phosphopantetheine mimetic


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.219 
  • R-Value Work: 0.190 
  • R-Value Observed: 0.191 

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This is version 1.5 of the entry. See complete history


Literature

Polyketide mimetics yield structural and mechanistic insights into product template domain function in nonreducing polyketide synthases.

Barajas, J.F.Shakya, G.Moreno, G.Rivera, H.Jackson, D.R.Topper, C.L.Vagstad, A.L.La Clair, J.J.Townsend, C.A.Burkart, M.D.Tsai, S.C.

(2017) Proc Natl Acad Sci U S A 114: E4142-E4148

  • DOI: https://doi.org/10.1073/pnas.1609001114
  • Primary Citation of Related Structures:  
    5KBZ

  • PubMed Abstract: 

    Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-β-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-β-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-β-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4'-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and the role of a protein-coordinated water network to selectively activate the C9 carbonyl for nucleophilic addition. The importance of the 4'-phosphate at the distal end of the pantetheine arm is demonstrated to both facilitate delivery of the heptaketide mimetic deep into the PT active site and anchor one end of this linear array to precisely meter C4 into close proximity to the catalytic His1345. Additional structural features, docking simulations, and mutational experiments characterize protein-substrate mimic interactions, which likely play roles in orienting and stabilizing interactions during the native multistep catalytic cycle. These findings afford a view of a polyketide "atom-replaced" mimetic in a NR-PKS active site that could prove general for other PKS domains.


  • Organizational Affiliation

    Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, CA 92697.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Noranthrone synthase
A, B
377Aspergillus parasiticusMutation(s): 0 
Gene Names: pksL1aflCpksA
EC: 2.3.1.221
UniProt
Find proteins for Q12053 (Aspergillus parasiticus (strain ATCC 56775 / NRRL 5862 / SRRC 143 / SU-1))
Explore Q12053 
Go to UniProtKB:  Q12053
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ12053
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
3B2
Query on 3B2

Download Ideal Coordinates CCD File 
C [auth A],
D [auth B]
(14R)-14-hydroxy-15,15-dimethyl-1-[5-({[(5-methyl-1,2-oxazol-3-yl)methyl]sulfanyl}methyl)-1,2-oxazol-3-yl]-4,9,13-trioxo-2-thia-5,8,12-triazahexadecan-16-yl dihydrogen phosphate
C23 H36 N5 O10 P S2
NHKWOPAZLQMZIQ-NRFANRHFSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.219 
  • R-Value Work: 0.190 
  • R-Value Observed: 0.191 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 89.374α = 90
b = 90.53β = 90
c = 90.836γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
HKL-2000data scaling
HKL-2000data reduction
PHENIXphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)United StatesGM 100738-02

Revision History  (Full details and data files)

  • Version 1.0: 2017-05-10
    Type: Initial release
  • Version 1.1: 2017-05-24
    Changes: Database references
  • Version 1.2: 2017-06-07
    Changes: Database references
  • Version 1.3: 2017-09-27
    Changes: Author supporting evidence
  • Version 1.4: 2019-12-25
    Changes: Author supporting evidence
  • Version 1.5: 2023-09-27
    Changes: Data collection, Database references, Refinement description