Journal: Nature / Year: 2014 Title: Structural rearrangements of a polyketide synthase module during its catalytic cycle. Authors: Jonathan R Whicher / Somnath Dutta / Douglas A Hansen / Wendi A Hale / Joseph A Chemler / Annie M Dosey / Alison R H Narayan / Kristina Håkansson / David H Sherman / Janet L Smith / Georgios Skiniotis / Abstract: The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical ...The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the β-keto intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.
History
Deposition
May 2, 2013
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Header (metadata) release
Jul 3, 2013
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Map release
Jun 25, 2014
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Update
Oct 22, 2014
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Current status
Oct 22, 2014
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_5663.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Reconstruction of the 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with thiophenol-pentaketide.
Voxel size
X=Y=Z: 2.24 Å
Density
Contour Level
By AUTHOR: 9.0 / Movie #1: 9
Minimum - Maximum
-32.860641479999998 - 67.041076660000002
Average (Standard dev.)
-0.03633139 (±1.48292053)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
192
192
192
Spacing
192
192
192
Cell
A=B=C: 430.08002 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
2.24
2.24
2.24
M x/y/z
192
192
192
origin x/y/z
0.000
0.000
0.000
length x/y/z
430.080
430.080
430.080
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-132
-122
-147
NX/NY/NZ
250
274
261
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
192
192
192
D min/max/mean
-32.861
67.041
-0.036
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Supplemental data
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Sample components
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Entire : The 5th module from the pikromycin biosynthetic pathway (PikAIII)...
Entire
Name: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with thiophenol-pentaketide
Components
Sample: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with thiophenol-pentaketide
Protein or peptide: PikAIII
Ligand: Thiophenol-pentaketide
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Supramolecule #1000: The 5th module from the pikromycin biosynthetic pathway (PikAIII)...
Supramolecule
Name: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with thiophenol-pentaketide type: sample / ID: 1000 Details: Sample was not frozen prior to loading on the grid. The sample was monodisperse. Oligomeric state: Dimer / Number unique components: 2
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 89 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1.5-2.0 seconds before plunging.
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Electron microscopy
Microscope
FEI TECNAI F20
Temperature
Min: 89 K / Max: 89 K
Alignment procedure
Legacy - Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification.
Date
Feb 2, 2012
Image recording
Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 269 / Average electron dose: 20 e/Å2
Electron beam
Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
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