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- PDB-9ijf: Structure of ATP-dependent diazotase CmaA6 -

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Basic information

Entry
Database: PDB / ID: 9ijf
TitleStructure of ATP-dependent diazotase CmaA6
ComponentsPutative AMP-binding enzyme
KeywordsLIGASE / diazotase
Function / homologyCoA-ligase activity / ANL, N-terminal domain / AMP-binding, conserved site / Putative AMP-binding domain signature. / AMP-dependent synthetase/ligase / AMP-binding enzyme / Putative AMP-binding enzyme
Function and homology information
Biological speciesKutzneria albida DSM 43870 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.73 Å
AuthorsKatsuyama, Y. / Kawai, S. / Ohnishi, Y.
Funding support Japan, 4items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)19H04645 Japan
Japan Society for the Promotion of Science (JSPS)19H05685 Japan
Japan Society for the Promotion of Science (JSPS)22H05130 Japan
Japan Society for the Promotion of Science (JSPS)22KJ1046 Japan
CitationJournal: Angew Chem Int Ed Engl / Year: 2025
Title: Structural Basis for the Catalytic Mechanism of ATP-Dependent Diazotase CmaA6.
Authors: Seiji Kawai / Masayuki Karasawa / Yoshitaka Moriwaki / Tohru Terada / Yohei Katsuyama / Yasuo Ohnishi /
Abstract: Although several diazotases have been recently reported, the details of the reaction mechanism are not yet understood. In this study, we investigated the mechanism of CmaA6, an ATP-dependent ...Although several diazotases have been recently reported, the details of the reaction mechanism are not yet understood. In this study, we investigated the mechanism of CmaA6, an ATP-dependent diazotase, which catalyzes the diazotization of 3-aminocoumaric acid using nitrous acid. X-ray crystallography and cryogenic electron microscopy-single particle analysis revealed CmaA6 structures in the substrate-free and AMP-binding states. Kinetic analysis suggested that CmaA6 catalyzes diazotization via a sequential reaction mechanism in which three substrates (nitrous acid, ATP, and 3-aminocoumaric acid) are simultaneously bound in the reaction pocket. The nitrous acid and 3-aminocoumaric acid binding sites were predicted based on the AMP-binding state and confirmed by site-directed mutagenesis. In addition, computational analysis revealed a tunnel for 3-aminocoumaric acid to enter the reaction pocket, which was advantageous for the sequential reaction mechanism. This study provides important insights into the catalytic mechanism of diazotization in natural product biosynthesis.
History
DepositionJun 22, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 7, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Putative AMP-binding enzyme
B: Putative AMP-binding enzyme
D: Putative AMP-binding enzyme
E: Putative AMP-binding enzyme
F: Putative AMP-binding enzyme
G: Putative AMP-binding enzyme
H: Putative AMP-binding enzyme
I: Putative AMP-binding enzyme


Theoretical massNumber of molelcules
Total (without water)496,3088
Polymers496,3088
Non-polymers00
Water81145
1
A: Putative AMP-binding enzyme
B: Putative AMP-binding enzyme
D: Putative AMP-binding enzyme
E: Putative AMP-binding enzyme


Theoretical massNumber of molelcules
Total (without water)248,1544
Polymers248,1544
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10070 Å2
ΔGint-46 kcal/mol
Surface area81400 Å2
MethodPISA
2
F: Putative AMP-binding enzyme
G: Putative AMP-binding enzyme
H: Putative AMP-binding enzyme
I: Putative AMP-binding enzyme


Theoretical massNumber of molelcules
Total (without water)248,1544
Polymers248,1544
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10720 Å2
ΔGint-43 kcal/mol
Surface area80290 Å2
MethodPISA
Unit cell
Length a, b, c (Å)94.163, 101.068, 157.411
Angle α, β, γ (deg.)76.310, 88.051, 62.283
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

#1: Protein
Putative AMP-binding enzyme


Mass: 62038.547 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Kutzneria albida DSM 43870 (bacteria) / Gene: KALB_2256 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: W5W4E6
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 45 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.82 Å3/Da / Density % sol: 56.4 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 200 mM magnesium formate dihydrate, 12% (w/v) PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-1A / Wavelength: 1.03 Å
DetectorType: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Jun 11, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03 Å / Relative weight: 1
ReflectionResolution: 2.73→152.34 Å / Num. obs: 130402 / % possible obs: 98.7 % / Redundancy: 3.7 % / CC1/2: 0.972 / Net I/σ(I): 18.8
Reflection shellResolution: 2.73→2.78 Å / Redundancy: 3.7 % / Mean I/σ(I) obs: 2.4 / Num. unique obs: 6508 / CC1/2: 0.764 / % possible all: 98.17

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Processing

Software
NameVersionClassification
PHENIX1.21.1_5286refinement
Coot1.20.1_4487model building
DIALSdata reduction
DIALSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: AlphaFold

Resolution: 2.73→65.95 Å / SU ML: 0.4119 / Cross valid method: FREE R-VALUE / σ(F): 1.97 / Phase error: 26.3754
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2514 1952 1.5 %
Rwork0.1943 128363 -
obs0.1952 130315 98.64 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 45.26 Å2
Refinement stepCycle: LAST / Resolution: 2.73→65.95 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms32072 0 0 45 32117
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.008532873
X-RAY DIFFRACTIONf_angle_d1.020644734
X-RAY DIFFRACTIONf_chiral_restr0.05414876
X-RAY DIFFRACTIONf_plane_restr0.00975920
X-RAY DIFFRACTIONf_dihedral_angle_d17.352411862
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.73-2.80.38791380.29439137X-RAY DIFFRACTION98.22
2.8-2.870.38491370.27599131X-RAY DIFFRACTION98.22
2.87-2.960.36921500.2539168X-RAY DIFFRACTION98.41
2.96-3.050.28311510.24749131X-RAY DIFFRACTION98.46
3.05-3.160.33011520.24139156X-RAY DIFFRACTION98.56
3.16-3.290.26861360.24569111X-RAY DIFFRACTION98.61
3.29-3.440.33441480.23559155X-RAY DIFFRACTION98.66
3.44-3.620.26191200.20619264X-RAY DIFFRACTION98.77
3.62-3.850.25851420.18589131X-RAY DIFFRACTION98.83
3.85-4.140.24251520.18569178X-RAY DIFFRACTION98.82
4.14-4.560.18831210.15449205X-RAY DIFFRACTION98.81
4.56-5.220.2071400.15019114X-RAY DIFFRACTION97.89
5.22-6.580.21321250.17719244X-RAY DIFFRACTION99.54
6.58-65.950.18051400.15149238X-RAY DIFFRACTION99.16

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