PDB-6cxt: Crystal structure of FAD-dependent dehydrogenase

Basic information

• Entry: Database: PDB / ID: 6cxt
• Title: Crystal structure of FAD-dependent dehydrogenase

Components

• Alpha/beta hydrolase fold protein
• Butyryl-CoA dehydrogenase

• Keywords: OXIDOREDUCTASE / acyl carrier protein / FAD-dependent enzyme / natural product biosynthesis

Function / homology

Function:

short-chain acyl-CoA dehydrogenase / short-chain fatty acyl-CoA dehydrogenase activity / flavin adenine dinucleotide binding / hydrolase activity

Domain/homology:

Butyryl-Coa Dehydrogenase, subunit A; domain 1 / Acyl-CoA dehydrogenase/oxidase, N-terminal domain / Butyryl-CoA Dehydrogenase, subunit A, domain 2 / Butyryl-CoA Dehydrogenase, subunit A; domain 2 / Acyl-CoA dehydrogenase/oxidase C-terminal / Acyl-CoA dehydrogenase/oxidase, N-terminal / Acyl-CoA dehydrogenase, N-terminal domain / Acyl-CoA dehydrogenase, C-terminal domain / Acyl-CoA oxidase/dehydrogenase, middle domain / Acyl-CoA dehydrogenase, middle domain / Acyl-CoA dehydrogenase/oxidase, N-terminal domain superfamily / Butyryl-CoA Dehydrogenase, subunit A, domain 3 / : / Acyl-CoA oxidase/dehydrogenase, middle domain superfamily / Acyl-CoA dehydrogenase/oxidase, N-terminal and middle domain superfamily / Acyl-CoA dehydrogenase-like, C-terminal / Alpha/beta hydrolase family / Butyryl-CoA Dehydrogenase, subunit A; domain 3 / Alpha/beta hydrolase fold-1 / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold / Up-down Bundle / Beta Barrel / Orthogonal Bundle / Mainly Beta / Mainly Alpha

Component:

FLAVIN-ADENINE DINUCLEOTIDE / Chem-FK4 / Alpha/beta hydrolase fold protein / Butyryl-CoA dehydrogenase

• Biological species: Marinomonas mediterranea (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
• Authors: Agarwal, V.

Funding support

United States, 1items

Organization	Grant number	Country
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)	R00ES026620	United States

• Citation: Journal: Biochemistry / Year: 2019; Title: Insights into Thiotemplated Pyrrole Biosynthesis Gained from the Crystal Structure of Flavin-Dependent Oxidase in Complex with Carrier Protein.; Authors: Thapa, H.R. / Robbins, J.M. / Moore, B.S. / Agarwal, V.

History

Deposition	Apr 4, 2018	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Jan 16, 2019	Provider: repository / Type: Initial release
Revision 1.1	Mar 6, 2019	Group: Data collection / Database references Category: citation / database_PDB_rev / database_PDB_rev_record / pdbx_database_proc Item: _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.2	Dec 18, 2019	Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3	Oct 4, 2023	Group: Data collection / Database references / Refinement description Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / refine_hist Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine_hist.d_res_low
Revision 1.4	Nov 13, 2024	Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

Assembly

Deposited unit

A: Alpha/beta hydrolase fold protein

B: Butyryl-CoA dehydrogenase

hetero molecules

Summary:

• 51.6 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	51,642	6
Polymers	50,297	2
Non-polymers	1,345	4
Water	6,215	345

1

A: Alpha/beta hydrolase fold protein

B: Butyryl-CoA dehydrogenase

hetero molecules

A: Alpha/beta hydrolase fold protein

B: Butyryl-CoA dehydrogenase

hetero molecules

A: Alpha/beta hydrolase fold protein

B: Butyryl-CoA dehydrogenase

hetero molecules

A: Alpha/beta hydrolase fold protein

B: Butyryl-CoA dehydrogenase

hetero molecules

Summary:

• defined by author&software
• Evidence: homology
• 207 kDa, 8 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	206,567	24
Polymers	201,187	8
Non-polymers	5,380	16
Water	144	8

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	4_665	-x+1,-y+1,z	1
crystal symmetry operation	7_556	y,x,-z+4/3	1
crystal symmetry operation	10_666	-y+1,-x+1,-z+4/3	1

Calculated values:

Buried area	34200 Å2
ΔGint	-149 kcal/mol
Surface area	59830 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	104.416, 104.416, 234.139
Angle α, β, γ (deg.)	90.00, 90.00, 120.00
Int Tables number	181
Space group name H-M	P6422

Components on special symmetry positions

ID	Model	Components
1	1	A-230- HOH

Components

Protein , 2 types, 2 molecules A B

#1: Protein

A Alpha/beta hydrolase fold protein / Bmp1(CP)

Details:

Mass: 8592.757 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Marinomonas mediterranea (strain ATCC 700492 / JCM 21426 / NBRC 103028 / MMB-1) (bacteria)
Strain: ATCC 700492 / JCM 21426 / NBRC 103028 / MMB-1 / Gene: Marme_4088 / Production host: Escherichia coli (E. coli) / References: UniProt: F2K074

#2: Protein

B Butyryl-CoA dehydrogenase / Bmp3

Details:

Mass: 41703.918 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Marinomonas mediterranea (strain ATCC 700492 / JCM 21426 / NBRC 103028 / MMB-1) (bacteria)
Strain: ATCC 700492 / JCM 21426 / NBRC 103028 / MMB-1 / Gene: Marme_4091 / Production host: Escherichia coli (E. coli)
References: UniProt: F2K077, short-chain acyl-CoA dehydrogenase

Non-polymers , 4 types, 349 molecules

#3: Chemical

A-100 ChemComp-FK4 / S-[2-({N-[(2R)-2-hydroxy-4-{[(R)-hydroxy(oxo)-lambda~5~-phosphanyl]oxy}-3,3-dimethylbutanoyl]-beta-alanyl}amino)ethyl] 1H-pyrrole-2-carbothioate

Details:

Mass: 435.432 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₁₆H₂₆N₃O₇PS / Feature type: SUBJECT OF INVESTIGATION

#4: Chemical

B-401 ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE

Details:

Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂₇H₃₃N₉O₁₅P₂ / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM

#5: Chemical

B-402 B-403 ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL

Details:

Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₂H₆O₂

#6: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 345 / Source method: isolated from a natural source / Formula: H₂O

Details

• Has protein modification: Y

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION / Number of used crystals: 1

Sample preparation

• Crystal: Density Matthews: 3.66 ų/Da / Density % sol: 66.42 %
• Crystal grow: Temperature: 279 K / Method: vapor diffusion, hanging drop / pH: 6 ; Details: 15 mM magnesium acetate, 50 mM sodium cacodylate, pH 6.0, 1.8 M ammonium sulfate

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: ALS / Beamline: 8.2.1 / Wavelength: 1 Å
• Detector: Type: ADSC QUANTUM 315r / Detector: CCD / Date: Nov 7, 2016
• Radiation: Monochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1 Å / Relative weight: 1
• Reflection: Resolution: 1.9→45 Å / Num. obs: 60078 / % possible obs: 100 % / Redundancy: 23.1 % / R_merge(I) obs: 0.1 / R_pim(I) all: 0.021 / R_rim(I) all: 0.102 / Χ²: 1.357 / Net I/σ(I): 7.7 / Num. measured all: 1386667

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	Rmerge(I) obs	Num. unique obs	CC1/2	Rpim(I) all	Rrim(I) all	Χ2	% possible all
1.9-1.93	22.2	0.645	2932	0.971	0.139	0.66	0.467	100
1.93-1.97	22.9	0.546	2950	0.977	0.116	0.558	0.473	100
1.97-2.01	23.2	0.45	2948	0.991	0.095	0.46	0.502	100
2.01-2.05	23.4	0.381	2928	0.987	0.08	0.39	0.542	100
2.05-2.09	23.4	0.321	2945	0.99	0.067	0.328	0.596	100
2.09-2.14	23.5	0.266	2950	0.993	0.056	0.272	0.646	100
2.14-2.19	23.5	0.232	2960	0.994	0.049	0.237	0.71	100
2.19-2.25	23.5	0.22	2957	0.994	0.046	0.225	0.77	100
2.25-2.32	23.5	0.19	2973	0.996	0.04	0.194	0.815	100
2.32-2.39	23.6	0.169	2969	0.997	0.035	0.172	0.882	100
2.39-2.48	23.7	0.148	2965	0.997	0.031	0.151	1.01	100
2.48-2.58	23.7	0.137	2975	0.997	0.029	0.14	1.116	100
2.58-2.7	23.7	0.124	3000	0.998	0.026	0.127	1.29	100
2.7-2.84	23.6	0.113	2989	0.998	0.024	0.116	1.528	100
2.84-3.02	23.5	0.109	3023	0.997	0.023	0.111	1.891	100
3.02-3.25	23.1	0.096	3023	0.998	0.02	0.098	2.481	100
3.25-3.58	22.5	0.091	3038	0.997	0.02	0.093	3.221	100
3.58-4.09	21.7	0.076	3072	0.999	0.017	0.078	3.425	100
4.09-5.15	22.3	0.062	3128	0.999	0.013	0.064	2.908	99.9
5.15-45	21.6	0.043	3353	0.999	0.01	0.044	1.834	99.8

Processing

Software

Name	Version	Classification
REFMAC	5.8.0158	refinement
HKL-2000		data reduction
HKL-2000		data scaling
PDB_EXTRACT	3.24	data extraction
PHASER		phasing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entries 4IRN & 4ETW

4irn

4etw

Resolution: 1.9→45 Å / Cor.coef. F_o:F_c: 0.957 / Cor.coef. F_o:F_c free: 0.945 / SU B: 2.497 / SU ML: 0.073 / Cross valid method: THROUGHOUT / ESU R: 0.109 / ESU R Free: 0.108 / Stereochemistry target values: MAXIMUM LIKELIHOOD

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.21072	1933	3.3 %	RANDOM
Rwork	0.18184	-	-	-
obs	0.1828	56340	96.91 %	-

• Solvent computation: Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK

Displacement parameters

B_iso mean: 30.042 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	0 Å2	0 Å2	-0 Å2
2-	-	0 Å2	0 Å2
3-	-	-	-0 Å2

Refinement step

Cycle: 1 / Resolution: 1.9→45 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	3415	0	89	345	3849

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.018	0.019	3611
X-RAY DIFFRACTION	r_bond_other_d
X-RAY DIFFRACTION	r_angle_refined_deg	1.881	1.977	4897
X-RAY DIFFRACTION	r_angle_other_deg
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.283	5	449
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	37.73	24.643	168
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	14.196	15	585
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	19.58	15	18
X-RAY DIFFRACTION	r_chiral_restr	0.143	0.2	539
X-RAY DIFFRACTION	r_gen_planes_refined	0.01	0.02	2740
X-RAY DIFFRACTION	r_gen_planes_other
X-RAY DIFFRACTION	r_nbd_refined
X-RAY DIFFRACTION	r_nbd_other
X-RAY DIFFRACTION	r_nbtor_refined
X-RAY DIFFRACTION	r_nbtor_other
X-RAY DIFFRACTION	r_xyhbond_nbd_refined
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined
X-RAY DIFFRACTION	r_symmetry_vdw_other
X-RAY DIFFRACTION	r_symmetry_hbond_refined
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	2.794	2.709	1781
X-RAY DIFFRACTION	r_mcbond_other
X-RAY DIFFRACTION	r_mcangle_it	3.938	4.039	2223
X-RAY DIFFRACTION	r_mcangle_other
X-RAY DIFFRACTION	r_scbond_it	3.496	3.034	1826
X-RAY DIFFRACTION	r_scbond_other
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other
X-RAY DIFFRACTION	r_long_range_B_refined	7.642	38.285	5955
X-RAY DIFFRACTION	r_long_range_B_other
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 1.901→1.95 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.289	128	-
Rwork	0.223	3708	-
obs	-	-	88.55 %