PDB-3fdy: Pyranose 2-oxidase thermostable triple mutant, T169G/E542K/V546C

Basic information

• Entry: Database: PDB / ID: 3fdy
• Title: Pyranose 2-oxidase thermostable triple mutant, T169G/E542K/V546C
• Components: Pyranose oxidase (Pyranose 2-oxidase)
• Keywords: OXIDOREDUCTASE / pyranose oxidase / thermostable / mutant / GMC oxidoreductase / 8-ALPHA-(N3) HISTIDYL flavinylation

Function / homology

Function:

pyranose oxidase / pyranose oxidase activity / flavin adenine dinucleotide binding / periplasmic space

Domain/homology:

Phage tail proteins - 2 layer sandwich fold - #50 / Pyranose 2-oxidase / Phage tail proteins - 2 layer sandwich fold / : / Glucose-methanol-choline oxidoreductase, C-terminal / GMC oxidoreductase / FAD/NAD(P)-binding domain superfamily / 2-Layer Sandwich / Alpha Beta

Component:

FLAVIN-ADENINE DINUCLEOTIDE / Pyranose 2-oxidase

• Biological species: Trametes multicolor (fungus)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.55 Å
• Authors: Tan, T.C. / Divne, C.
• Citation: Journal: Biotechnol J / Year: 2009; Title: A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications; Authors: Spadiut, O. / Radakovits, K. / Pisanelli, I. / Salaheddin, C. / Yamabhai, M. / Tan, T.-C. / Divne, C. / Haltrich, D.

History

Deposition	Nov 26, 2008	Deposition site: RCSB / Processing site: PDBJ
Revision 1.0	Mar 31, 2009	Provider: repository / Type: Initial release
Revision 1.1	Jul 13, 2011	Group: Advisory / Version format compliance
Revision 1.2	May 23, 2018	Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.3	Nov 10, 2021	Group: Database references / Derived calculations Category: database_2 / struct_conn / struct_ref_seq_dif / struct_site Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4	Nov 1, 2023	Group: Data collection / Refinement description Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.5	Oct 30, 2024	Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

Assembly

Deposited unit

A: Pyranose oxidase (Pyranose 2-oxidase)

hetero molecules

Summary:

• 70.4 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	70,355	3
Polymers	69,374	1
Non-polymers	981	2
Water	8,467	470

1

A: Pyranose oxidase (Pyranose 2-oxidase)

hetero molecules

A: Pyranose oxidase (Pyranose 2-oxidase)

hetero molecules

A: Pyranose oxidase (Pyranose 2-oxidase)

hetero molecules

A: Pyranose oxidase (Pyranose 2-oxidase)

hetero molecules

Summary:

• defined by author&software
• 281 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	281,418	12
Polymers	277,495	4
Non-polymers	3,923	8
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_665	-x+1,-y+1,z	1
crystal symmetry operation	7_556	y,x,-z+1	1
crystal symmetry operation	8_666	-y+1,-x+1,-z+1	1

Calculated values:

Buried area	24490 Å2
ΔGint	-125 kcal/mol
Surface area	80830 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	101.580, 101.580, 126.850
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	94
Space group name H-M	P42212

Components

#1: Protein

A Pyranose oxidase (Pyranose 2-oxidase)

Details:

Mass: 69373.805 Da / Num. of mol.: 1 / Mutation: T169G, E542K, V546C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trametes multicolor (fungus) / Plasmid: PET21(D+) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q7ZA32, pyranose oxidase

#2: Chemical

A-801 ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE

Details:

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

#3: Chemical

A-901 ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID

Details:

Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₆H₁₃NO₄S / Comment: pH buffer*YM

#4: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 470 / Source method: isolated from a natural source / Formula: H₂O

• Has protein modification: Y

Experimental details

Experiment

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

Sample preparation

• Crystal: Density Matthews: 2.36 ų/Da / Density % sol: 47.85 %
• Crystal grow: Temperature: 298 K / Method: vapor diffusion, hanging drop / pH: 5.2 ; Details: 10% (w/v) monomethylether PEG 2000, 0.1M Mes pH 5.2, 50mM MgCl2, 25% glycerol, VAPOR DIFFUSION, HANGING DROP, temperature 298K

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: MAX II / Beamline: I911-3 / Wavelength: 1 Å
• Detector: Type: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jun 8, 2008 / Details: mirrors
• Radiation: Monochromator: Double crystal 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: 1.55→79.3 Å / Num. obs: 1581896 / % possible obs: 99.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 16.4 % / R_sym value: 0.159 / Net I/σ(I): 12.8
• Reflection shell: Resolution: 1.55→1.6 Å / Redundancy: 11.3 % / Mean I/σ(I) obs: 2.8 / Num. unique all: 96227 / R_sym value: 0.885 / % possible all: 97.5

Processing

Software

Name	Version	Classification
AMoRE		phasing
REFMAC	5.2.0019	refinement
XDS		data reduction
XDS		data scaling

Refinement

Method to determine structure: FOURIER SYNTHESIS
Starting model: PDB ENTRY 2IGO

2igo

Resolution: 1.55→53.8 Å / Cor.coef. F_o:F_c: 0.952 / Cor.coef. F_o:F_c free: 0.936 / SU B: 3.399 / SU ML: 0.064 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / ESU R: 0.077 / ESU R Free: 0.08 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.21883	2899	3 %	RANDOM
Rwork	0.18602	-	-	-
obs	0.18703	93323	99.77 %	-

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

Displacement parameters

B_iso mean: 9.57 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	-0.18 Å2	0 Å2	0 Å2
2-	-	-0.18 Å2	0 Å2
3-	-	-	0.35 Å2

Refinement step

Cycle: LAST / Resolution: 1.55→53.8 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4538	0	65	470	5073

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.022	0.022	4724
X-RAY DIFFRACTION	r_bond_other_d	0.002	0.02	3188
X-RAY DIFFRACTION	r_angle_refined_deg	1.981	1.963	6423
X-RAY DIFFRACTION	r_angle_other_deg	1.054	3	7765
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.945	5	575
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	37.119	24.464	224
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	13.86	15	760
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	14.412	15	27
X-RAY DIFFRACTION	r_chiral_restr	0.208	0.2	696
X-RAY DIFFRACTION	r_gen_planes_refined	0.009	0.02	5239
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	946
X-RAY DIFFRACTION	r_nbd_refined	0.222	0.2	974
X-RAY DIFFRACTION	r_nbd_other	0.215	0.2	3475
X-RAY DIFFRACTION	r_nbtor_refined	0.192	0.2	2351
X-RAY DIFFRACTION	r_nbtor_other	0.097	0.2	2561
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.168	0.2	333
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	0.2	0.2	13
X-RAY DIFFRACTION	r_symmetry_vdw_other	0.223	0.2	71
X-RAY DIFFRACTION	r_symmetry_hbond_refined	0.146	0.2	21
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	1.4	1.5	3589
X-RAY DIFFRACTION	r_mcbond_other	0.366	1.5	1153
X-RAY DIFFRACTION	r_mcangle_it	1.698	2	4673
X-RAY DIFFRACTION	r_scbond_it	2.955	3	2115
X-RAY DIFFRACTION	r_scangle_it	4.069	4.5	1750
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 1.55→1.634 Å / Total num. of bins used: 10

	Rfactor	Num. reflection	% reflection
Rfree	0.311	399	-
Rwork	0.265	13283	-
obs	-	-	98.45 %

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°2)	L12 (°2)	L13 (°2)	L22 (°2)	L23 (°2)	L33 (°2)	S11 (Å °)	S12 (Å °)	S13 (Å °)	S21 (Å °)	S22 (Å °)	S23 (Å °)	S31 (Å °)	S32 (Å °)	S33 (Å °)	T11 (Å2)	T12 (Å2)	T13 (Å2)	T22 (Å2)	T23 (Å2)	T33 (Å2)	Origin x (Å)	Origin y (Å)	Origin z (Å)
1	13.7579	1.3763	-6.1903	0.6444	-1.9049	6.0471	-0.0599	0.673	-0.9136	0.0142	-0.1039	-0.1335	0.343	-0.4536	0.1638	0.0584	-0.0225	0.0062	0.0475	-0.0844	0.0254	70.2711	40.3169	13.352
2	0.3731	-0.1136	0.012	0.3031	-0.131	0.0608	0.0134	0.0528	0.0033	-0.0058	0.0101	0.0565	0.0007	-0.0058	-0.0235	0.0209	0.0004	0.0075	0.0381	-0.004	-0.011	66.7523	50.73	22.3974
3	0.102	-0.0032	0.0022	0.0582	-0.0207	0.0074	-0.0153	-0.0071	0.0016	0.0226	0.0023	-0.0137	-0.0181	0.0035	0.0129	0.0314	-0.0025	0.0063	0.019	0.0018	0.0029	54.9788	44.4225	48.8733
4	0.0643	-0.0474	0.1282	0.2216	-0.2391	0.3678	-0.0122	0.0047	0.0021	-0.0015	-0.0092	-0.0117	0.009	-0.0079	0.0214	0.0189	0.0046	0.0095	0.0233	-0.0001	0.0036	66.8701	46.4055	44.1709
5	1.7404	-0.0439	0.1791	0.3302	-0.2737	0.9688	0.0247	0.0177	0.0371	-0.0214	-0.0416	-0.0302	-0.0202	0.0857	0.0168	0.0223	-0.0176	0.02	0.0065	0.0064	0.0057	82.8375	66.5331	33.0526
6	0.6107	-0.2685	-0.4544	0.5413	0.7819	1.1386	0.0398	0.0375	0.0418	-0.0108	-0.0553	-0.0279	0.0089	-0.0959	0.0155	0.0279	0.0029	0.0074	0.0074	0.0157	0.0013	61.6526	75.5422	39.15
7	5.2509	-3.2444	0.5271	3.5262	1.7041	2.7606	-0.0794	-0.0817	0.2631	-0.0955	0.1767	-0.1793	-0.1903	0.0337	-0.0973	0.021	-0.0338	-0.0103	-0.0113	0.0085	0.0155	71.9783	81.1164	52.1015
8	0.0748	-0.1888	-0.1593	0.4914	0.3821	0.365	0.0034	0.0491	0.009	-0.0274	-0.0351	0.0424	-0.0394	0.0086	0.0317	0.0223	0.0031	0.009	0.0153	0.0088	0.0107	60.1385	66.4001	31.0671
9	0.5322	0.1043	-0.3502	0.0407	-0.0489	0.2495	0.0267	0.1419	-0.0341	-0.0163	0.0155	0.0624	0.0264	-0.0377	-0.0423	0.0249	0.0011	-0.0003	0.0381	-0.0111	-0.0127	64.5561	47.7143	16.4615
10	1.7573	-0.4854	-0.6103	0.1951	0.3712	0.8854	0.0447	0.1012	-0.0463	-0.0102	-0.0739	-0.0051	0.0277	-0.0125	0.0292	0.0253	0.0101	0.034	0.0157	-0.0248	0.0167	82.411	35.7906	23.3414
11	12.7008	-3.2994	-3.7317	4.2483	0.4686	1.1704	0.097	0.5413	-0.4635	-0.0678	-0.0192	0.0041	0.1462	-0.3035	-0.0778	0.0262	-0.0218	-0.0231	-0.0186	-0.054	0.0262	63.7961	32.4239	23.9803
12	0.037	0.0739	-0.0537	0.1792	0.0097	0.5121	-0.0097	0.0033	-0.0327	-0.0153	-0.0234	-0.0059	0.0421	0.08	0.0332	0.017	0.0145	0.0154	0.0199	-0.0014	0.0163	80.5565	40.1274	35.51
13	0.7682	-0.5425	0.1257	0.7941	0.0228	0.0509	-0.0686	-0.0056	0.1144	0.022	-0.0002	-0.0591	0.0362	-0.0214	0.0687	0.0119	0.0138	-0.0111	0.0211	0.0067	0.0129	56.4731	77.3969	53.6219
14	7.1185	-1.9956	-1.1408	1.7087	-1.139	2.0347	-0.0928	0.2182	0.9988	0.2349	0.0909	-0.3854	-0.0497	-0.2098	0.0019	-0.0462	0.0157	-0.0571	-0.0659	0.025	0.115	46.0942	92.7171	47.5377
15	1.1825	-0.5899	-0.0567	0.308	-0.0235	0.1984	-0.0484	0.0701	0.0059	-0.0021	0.009	-0.0025	0.002	-0.0249	0.0394	0.0099	-0.0001	-0.0087	0.016	0.0072	0.0066	56.1235	76.6588	46.642
16	0.45	1.5635	0.0848	5.4322	0.2947	0.016	-0.6846	-0.7618	-0.1171	-0.6437	0.8186	0.2601	1.1174	1.0195	-0.134	0.1259	0.0028	-0.0052	0.1289	-0.0003	0.1299	51.4973	61.3299	52.5396
17	0.4119	-0.1118	-0.1921	0.0325	0.0523	0.0896	-0.0113	0.034	0.0269	-0.0107	0.0189	0.0002	0.0295	-0.0043	-0.0076	0.0188	0.0069	0.0045	0.0314	-0.0002	0.0045	59.8939	66.9117	51.859
18	0.9014	0.5852	0.7097	1.1021	0.4452	0.5591	0.0212	-0.0067	-0.0806	0.0236	0.0241	-0.0305	0.0037	0.0032	-0.0452	0.0101	-0.0003	0.0246	0.0183	0.0064	0.0082	75.5255	39.7708	45.3236
19	0.4	-0.2213	-0.1166	0.1466	0.1344	0.2357	-0.0259	-0.0129	-0.001	0.0217	0.0164	-0.002	0.01	0.0069	0.0095	0.0196	0.004	0.0045	0.0222	0.004	0.0042	69.6402	63.1379	57.36
20	0.1765	-0.0483	-0.1513	0.1838	0.0463	0.2833	-0.0096	0.0043	-0.0125	-0.0057	0.0008	-0.0344	-0.0161	0.0049	0.0088	0.0143	-0.0018	0.0154	0.0201	-0.001	0.0095	81.8031	52.7123	29.7754

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	A	43 - 48
2	X-RAY DIFFRACTION	2	A	49 - 86
3	X-RAY DIFFRACTION	3	A	87 - 140
4	X-RAY DIFFRACTION	4	A	141 - 171
5	X-RAY DIFFRACTION	5	A	172 - 204
6	X-RAY DIFFRACTION	6	A	205 - 225
7	X-RAY DIFFRACTION	7	A	226 - 235
8	X-RAY DIFFRACTION	8	A	236 - 263
9	X-RAY DIFFRACTION	9	A	264 - 285
10	X-RAY DIFFRACTION	10	A	286 - 299
11	X-RAY DIFFRACTION	11	A	300 - 310
12	X-RAY DIFFRACTION	12	A	311 - 357
13	X-RAY DIFFRACTION	13	A	358 - 383
14	X-RAY DIFFRACTION	14	A	384 - 407
15	X-RAY DIFFRACTION	15	A	408 - 451
16	X-RAY DIFFRACTION	16	A	452 - 460
17	X-RAY DIFFRACTION	17	A	461 - 477
18	X-RAY DIFFRACTION	18	A	478 - 505
19	X-RAY DIFFRACTION	19	A	506 - 545
20	X-RAY DIFFRACTION	20	A	546 - 618