Japan Agency for Medical Research and Development (AMED)
JP20am0101071
Japan
Citation
Journal: Angew Chem Int Ed Engl / Year: 2021 Title: Structural and Functional Analyses of the Tridomain-Nonribosomal Peptide Synthetase FmoA3 for 4-Methyloxazoline Ring Formation. Authors: Yohei Katsuyama / Kaoru Sone / Ayaka Harada / Seiji Kawai / Naoki Urano / Naruhiko Adachi / Toshio Moriya / Masato Kawasaki / Kazuo Shin-Ya / Toshiya Senda / Yasuo Ohnishi / Abstract: Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and ...Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α-methyl-l-serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography (apo-form, with adenylyl-imidodiphosphate and α-methyl-l-seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α-methyl-l-serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.
History
Deposition
Aug 11, 2020
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Header (metadata) release
Apr 14, 2021
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Map release
Apr 14, 2021
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Update
Jun 30, 2021
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Current status
Jun 30, 2021
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
EMPIAR-11059 (Title: Structural and functional analyses of the tridomain-nonribosomal peptide synthetase FmoA3 for 4-methyloxazoline ring formation Data size: 1.4 TB Data #1: Structural and functional analyses of the tridomain-nonribosomal peptide synthetase FmoA3 for 4-methyloxazoline ring formation [micrographs - multiframe])
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: The grid was washed by acetone prior to use.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 15 seconds (blot force 25).
Details
This sample was mono-disperse.
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Electron microscopy
Microscope
FEI TALOS ARCTICA
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1886 / Average exposure time: 50.07 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Model: Talos Arctica / Image courtesy: FEI Company
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Image processing
Particle selection
Number selected: 819697
CTF correction
Software - Name: Gctf (ver. 1.06)
Startup model
Type of model: OTHER Details: An ab initio model was generated using RELION3's own implementation of Stochastic Gradient Descent (SGD) algorithm and low-pass filtered to 60 A for use as an initial model for 3D classification.
Final reconstruction
Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 76171
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)
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