PDB-4ub9: Structural and catalytic characterization of molinate hydrolase

Basic information

• Entry: Database: PDB / ID: 4ub9
• Title: Structural and catalytic characterization of molinate hydrolase
• Components: Molinate hydrolase
• Keywords: HYDROLASE / amidohydrolase molinate thiocarbamate

Function / homology

Function:

hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds / metal ion binding

Domain/homology:

: / Urease, subunit C; domain 1 / Urease, subunit C, domain 1 / Amidohydrolase family / Metal-dependent hydrolase, composite domain superfamily / Amidohydrolase-related / Metal-dependent hydrolases / Metal-dependent hydrolase / Roll / TIM Barrel / Alpha-Beta Barrel / Mainly Beta / Alpha Beta

Component:

Molinate hydrolase

• Biological species: Gulosibacter molinativorax (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.27 Å
• Authors: Leite, J.P. / Duarte, M. / Paiva, A. / Ferreira-da-Silva, F. / Matias, P.M. / Nunes, O. / Gales, L.

Funding support

Portugal, 1items

Organization	Grant number	Country
FCT	FCOMP-01-0124-FEDER-027883 (PTDC/AAGTEC/3909/2012FCOMP)	Portugal

• Citation: Journal: Plos One / Year: 2015; Title: Structure-guided engineering of molinate hydrolase for the degradation of thiocarbamate pesticides.; Authors: Leite, J.P. / Duarte, M. / Paiva, A.M. / Ferreira-da-Silva, F. / Matias, P.M. / Nunes, O.C. / Gales, L.

History

Deposition	Aug 12, 2014	Deposition site: RCSB / Processing site: PDBE
Revision 1.0	Jun 24, 2015	Provider: repository / Type: Initial release
Revision 1.1	Feb 20, 2019	Group: Advisory / Data collection / Derived calculations Category: pdbx_data_processing_status / pdbx_validate_close_contact / struct_conn / struct_conn_type
Revision 1.2	May 1, 2024	Group: Data collection / Database references / Derived calculations / Refinement description Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ncs_dom_lim Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

Assembly

Deposited unit

A: Molinate hydrolase

B: Molinate hydrolase

C: Molinate hydrolase

D: Molinate hydrolase

E: Molinate hydrolase

F: Molinate hydrolase

G: Molinate hydrolase

H: Molinate hydrolase

hetero molecules

Summary:

• 438 kDa, 8 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	437,956	16
Polymers	437,432	8
Non-polymers	523	8
Water	10,845	602

1

A: Molinate hydrolase

B: Molinate hydrolase

C: Molinate hydrolase

D: Molinate hydrolase

hetero molecules

Summary:

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

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	218,978	8
Polymers	218,716	4
Non-polymers	262	4
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	12300 Å2
ΔGint	-176 kcal/mol
Surface area	59780 Å2
Method	PISA

2

E: Molinate hydrolase

hetero molecules

F: Molinate hydrolase

hetero molecules

G: Molinate hydrolase

H: Molinate hydrolase

hetero molecules

Summary:

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

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	218,978	8
Polymers	218,716	4
Non-polymers	262	4
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	4_558	-x+1/2,y+1/2,-z+3	1
crystal symmetry operation	3_455	x-1/2,y+1/2,z	1

Calculated values:

Buried area	12300 Å2
ΔGint	-176 kcal/mol
Surface area	59780 Å2
Method	PISA

3

E: Molinate hydrolase

hetero molecules

F: Molinate hydrolase

hetero molecules

G: Molinate hydrolase

H: Molinate hydrolase

hetero molecules

Summary:

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

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	218,978	8
Polymers	218,716	4
Non-polymers	262	4
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
crystal symmetry operation	3_545	x+1/2,y-1/2,z	1
crystal symmetry operation	2_658	-x+1,y,-z+3	1
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	12310 Å2
ΔGint	-171 kcal/mol
Surface area	59780 Å2
Method	PISA

4

F: Molinate hydrolase

hetero molecules

E: Molinate hydrolase

hetero molecules

G: Molinate hydrolase

H: Molinate hydrolase

hetero molecules

Summary:

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

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	218,978	8
Polymers	218,716	4
Non-polymers	262	4
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	4_548	-x+1/2,y-1/2,-z+3	1
crystal symmetry operation	2_658	-x+1,y,-z+3	1

Calculated values:

Buried area	12310 Å2
ΔGint	-171 kcal/mol
Surface area	59780 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	367.696, 99.079, 131.337
Angle α, β, γ (deg.)	90.00, 109.61, 90.00
Int Tables number	5
Space group name H-M	C121

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	A
2	1	B
1	2	A
2	2	C
1	3	A
2	3	D
1	4	A
2	4	E
1	5	A
2	5	F
1	6	A
2	6	G
1	7	A
2	7	H
1	8	B
2	8	C
1	9	B
2	9	D
1	10	B
2	10	E
1	11	B
2	11	F
1	12	B
2	12	G
1	13	B
2	13	H
1	14	C
2	14	D
1	15	C
2	15	E
1	16	C
2	16	F
1	17	C
2	17	G
1	18	C
2	18	H
1	19	D
2	19	E
1	20	D
2	20	F
1	21	D
2	21	G
1	22	D
2	22	H
1	23	E
2	23	F
1	24	E
2	24	G
1	25	E
2	25	H
1	26	F
2	26	G
1	27	F
2	27	H
1	28	G
2	28	H

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: PHE / Beg label comp-ID: PHE / End auth comp-ID: ASP / End label comp-ID: ASP / Refine code: _ / Auth seq-ID: 30 - 496 / Label seq-ID: 30 - 496

Dom-ID	Ens-ID	Auth asym-ID	Label asym-ID
1	1	A	A
2	1	B	B
1	2	A	A
2	2	C	C
1	3	A	A
2	3	D	D
1	4	A	A
2	4	E	E
1	5	A	A
2	5	F	F
1	6	A	A
2	6	G	G
1	7	A	A
2	7	H	H
1	8	B	B
2	8	C	C
1	9	B	B
2	9	D	D
1	10	B	B
2	10	E	E
1	11	B	B
2	11	F	F
1	12	B	B
2	12	G	G
1	13	B	B
2	13	H	H
1	14	C	C
2	14	D	D
1	15	C	C
2	15	E	E
1	16	C	C
2	16	F	F
1	17	C	C
2	17	G	G
1	18	C	C
2	18	H	H
1	19	D	D
2	19	E	E
1	20	D	D
2	20	F	F
1	21	D	D
2	21	G	G
1	22	D	D
2	22	H	H
1	23	E	E
2	23	F	F
1	24	E	E
2	24	G	G
1	25	E	E
2	25	H	H
1	26	F	F
2	26	G	G
1	27	F	F
2	27	H	H
1	28	G	G
2	28	H	H

NCS ensembles :

ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

Components

#1: Protein

A B C D E F G H
Molinate hydrolase

Details:

Mass: 54679.062 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gulosibacter molinativorax (bacteria) / Gene: molA / Production host: Escherichia coli (E. coli) / References: UniProt: G2XLB0

#2: Chemical

A-501 B-501 C-501 D-501 E-501 F-501 G-501 H-501
ChemComp-ZN / ZINC ION

Details:

Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn

#3: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 602 / Source method: isolated from a natural source / Formula: H₂O

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION

Sample preparation

• Crystal: Density Matthews: 2.58 ų/Da / Density % sol: 52.24 %
• Crystal grow: Temperature: 293 K / Method: vapor diffusion; Details: 0.095M sodium citrate tribasic dihydrate, pH 5.6-6.4, 19% v/v 2-propanol, 19% polyethylene glycol 4000, 5% v/v glycerol ZnCl2; PH range: 5.6 - 6.4

Data collection

Diffraction

ID	Mean temperature (K)	Crystal-ID
1	100	1
2	100	1

Diffraction source

Source	Site	Beamline	ID	Wavelength (Å)
SYNCHROTRON	ESRF	ID23-1	1	1.265
SYNCHROTRON	ESRF	ID14-4	2	0.9736, 2.13

Detector

Type	ID	Detector	Date
DECTRIS PILATUS 6M-F	1	PIXEL	Apr 14, 2014
ADSC QUANTUM 315	2	CCD	Dec 10, 2010

Radiation

ID	Protocol	Monochromatic (M) / Laue (L)	Scattering type	Wavelength-ID
1	SINGLE WAVELENGTH	M	x-ray	1
2	SINGLE WAVELENGTH	M	x-ray	2

Radiation wavelength

ID	Wavelength (Å)	Relative weight
1	1.265	1
2	0.9736	1
3	2.13	1

• Reflection: Resolution: 2.27→100 Å / Num. obs: 183046 / % possible obs: 93.6 % / Redundancy: 2.66 % / Net I/σ(I): 12.2

Processing

Software

Name	Version	Classification
REFMAC	5.8.0049	refinement
XDS		data reduction
SHARP		phasing
XSCALE		data scaling

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: inhouse structure of SeMet- mutant

Resolution: 2.27→123.72 Å / Cor.coef. F_o:F_c: 0.955 / Cor.coef. F_o:F_c free: 0.943 / SU B: 8.223 / SU ML: 0.183 / Cross valid method: THROUGHOUT / ESU R: 0.315 / ESU R Free: 0.215 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.22864	9639	5 %	RANDOM
Rwork	0.20095	-	-	-
obs	0.20234	183046	94.47 %	-

• 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: 49.423 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: 2.27→123.72 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	28776	0	8	602	29386

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.013	0.019	29384
X-RAY DIFFRACTION	r_bond_other_d	0.008	0.02	28112
X-RAY DIFFRACTION	r_angle_refined_deg	1.548	1.961	39912
X-RAY DIFFRACTION	r_angle_other_deg	1.297	3	64608
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.075	5	3728
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	34.986	23.818	1320
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	16.235	15	4848
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	22.334	15	224
X-RAY DIFFRACTION	r_chiral_restr	0.09	0.2	4520
X-RAY DIFFRACTION	r_gen_planes_refined	0.009	0.021	33384
X-RAY DIFFRACTION	r_gen_planes_other	0.006	0.02	6536
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	4.047	4.706	14936
X-RAY DIFFRACTION	r_mcbond_other	4.045	4.706	14935
X-RAY DIFFRACTION	r_mcangle_it	5.798	7.053	18656
X-RAY DIFFRACTION	r_mcangle_other	5.799	7.054	18657
X-RAY DIFFRACTION	r_scbond_it	4.596	5.171	14447
X-RAY DIFFRACTION	r_scbond_other	4.596	5.17	14445
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other	6.965	7.592	21255
X-RAY DIFFRACTION	r_long_range_B_refined	9.036	37.716	33504
X-RAY DIFFRACTION	r_long_range_B_other	9.029	37.707	33395
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-ID	Dom-ID	Auth asym-ID	Number	Rms dev position (Å)
1	1	A	30157	0.04
1	2	B	30157	0.04
2	1	A	30276	0.03
2	2	C	30276	0.03
3	1	A	30239	0.04
3	2	D	30239	0.04
4	1	A	30364	0.03
4	2	E	30364	0.03
5	1	A	30078	0.05
5	2	F	30078	0.05
6	1	A	30181	0.04
6	2	G	30181	0.04
7	1	A	30123	0.04
7	2	H	30123	0.04
8	1	B	30289	0.03
8	2	C	30289	0.03
9	1	B	30371	0.03
9	2	D	30371	0.03
10	1	B	30336	0.02
10	2	E	30336	0.02
11	1	B	30095	0.04
11	2	F	30095	0.04
12	1	B	30207	0.04
12	2	G	30207	0.04
13	1	B	30264	0.03
13	2	H	30264	0.03
14	1	C	30362	0.03
14	2	D	30362	0.03
15	1	C	30390	0.03
15	2	E	30390	0.03
16	1	C	30139	0.05
16	2	F	30139	0.05
17	1	C	30291	0.04
17	2	G	30291	0.04
18	1	C	30267	0.04
18	2	H	30267	0.04
19	1	D	30400	0.02
19	2	E	30400	0.02
20	1	D	30152	0.04
20	2	F	30152	0.04
21	1	D	30286	0.04
21	2	G	30286	0.04
22	1	D	30362	0.03
22	2	H	30362	0.03
23	1	E	30194	0.04
23	2	F	30194	0.04
24	1	E	30317	0.03
24	2	G	30317	0.03
25	1	E	30302	0.03
25	2	H	30302	0.03
26	1	F	30144	0.05
26	2	G	30144	0.05
27	1	F	30060	0.05
27	2	H	30060	0.05
28	1	G	30180	0.04
28	2	H	30180	0.04

LS refinement shell

Resolution: 2.275→2.334 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.379	498	-
Rwork	0.343	9839	-
obs	-	-	68.73 %