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- PDB-9npb: Crystal structure of the inactive conformation of a glycoside hyd... -

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Basic information

Entry
Database: PDB / ID: 9npb
TitleCrystal structure of the inactive conformation of a glycoside hydrolase (CapGH2b) from the GH2 family in the space group R3 at 2.45 A
ComponentsGlycoside hydrolase family 2
KeywordsHYDROLASE / Redox-regulation / glycosyl hydrolase / mannosidase / redox-switch / metagenome / disulfide bond
Biological speciesmetagenome (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.45 Å
AuthorsMartins, M.P. / Spadeto, J.P.M. / Miyamoto, R.Y. / Morais, M.A.B. / Murakami, M.T.
Funding support Brazil, 3items
OrganizationGrant numberCountry
Sao Paulo Research Foundation (FAPESP)2021/04891-3 Brazil
Sao Paulo Research Foundation (FAPESP)2021/09793-0 Brazil
Sao Paulo Research Foundation (FAPESP)2022/06298-0 Brazil
CitationJournal: Nat Commun / Year: 2026
Title: A disulfide redox switch mechanism regulates glycoside hydrolase function.
Authors: Marcele Pandeló Martins / Gustavo Henrique Martins / Felipe Jun Fuzita / João Paulo Menezes Spadeto / Renan Yuji Miyamoto / Felippe Mariano Colombari / Fabiane Stoffel / Luciano Graciani ...Authors: Marcele Pandeló Martins / Gustavo Henrique Martins / Felipe Jun Fuzita / João Paulo Menezes Spadeto / Renan Yuji Miyamoto / Felippe Mariano Colombari / Fabiane Stoffel / Luciano Graciani Dolce / Camila Ramos Dos Santos / Rodrigo Silva Araujo Streit / Antônio Carlos Borges / Rafael Henrique Galinari / Yoshihisa Yoshimi / Paul Dupree / Gabriela Felix Persinoti / Mariana Abrahão Bueno Morais / Mario Tyago Murakami /
Abstract: Disulfide bonds are a key post-translational modification involved in protein folding, structural stability, and functional regulation. Here, we demonstrate that a glycoside hydrolase from the GH2 ...Disulfide bonds are a key post-translational modification involved in protein folding, structural stability, and functional regulation. Here, we demonstrate that a glycoside hydrolase from the GH2 family undergoes reversible redox regulation through an intramolecular disulfide bond. The enzyme is inactive in its oxidized state and becomes active when reduced through a fully reversible process. Under oxidative conditions, multiple crystallographic and cryo-EM structures revealed a pronounced structural disorder in the active site, most prominent in the regulatory and catalytic loops, which disrupts the substrate binding site and, remarkably, the configuration of the acidic catalytic residues. Conversely, a high-resolution cryo-EM structure of the active (reduced) state unveiled a well-ordered active site with catalytic residues properly positioned for a classical Koshland retaining mechanism. This reversible order-disorder process based on a disulfide switch provides a mechanism for redox-dependent control of glycoside hydrolase activity, with potential implications for carbohydrate metabolism, microbial adaptation and biotechnological applications.
History
DepositionMar 11, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 21, 2026Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glycoside hydrolase family 2


Theoretical massNumber of molelcules
Total (without water)90,8171
Polymers90,8171
Non-polymers00
Water4,504250
1
A: Glycoside hydrolase family 2

A: Glycoside hydrolase family 2

A: Glycoside hydrolase family 2


Theoretical massNumber of molelcules
Total (without water)272,4503
Polymers272,4503
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area7680 Å2
ΔGint-40 kcal/mol
Surface area77430 Å2
MethodPISA
Unit cell
Length a, b, c (Å)180.437, 180.437, 69.827
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number146
Space group name H-MH3
Space group name HallR3

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Components

#1: Protein Glycoside hydrolase family 2


Mass: 90816.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: beta-mannosidase
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 250 / 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: 2.41 Å3/Da / Density % sol: 48.94 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop
Details: 10% PEG8000, 0.1 M Imidazole, pH 8, 2 mM Tris(2-carboxyethyl)phosphine

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: LNLS SIRIUS / Beamline: MANACA / Wavelength: 1.45864 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Nov 20, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.45864 Å / Relative weight: 1
ReflectionResolution: 2.45→45.11 Å / Num. obs: 31143 / % possible obs: 100 % / Redundancy: 10.35 % / CC1/2: 0.977 / Rmerge(I) obs: 0.161 / Rrim(I) all: 0.216 / Net I/σ(I): 3.14
Reflection shell
Resolution (Å)Rmerge(I) obsNum. unique obsCC1/2Rrim(I) allDiffraction-ID
2.45-2.61.15537490.211.5871
2.6-2.780.82234400.3411.1271
2.78-30.58931580.5120.8011
3-3.280.32727850.7830.4451
3.28-3.670.18124990.9180.2441
3.67-4.230.12120880.9610.1611
4.23-5.170.08818500.9810.1151
5.17-7.260.09113850.9820.1171
7.26-45.110.0477160.9940.061

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Processing

Software
NameVersionClassification
PHENIX1.21.2_5419refinement
XDSdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.45→45.11 Å / SU ML: 0.3649 / Cross valid method: FREE R-VALUE / σ(F): 1.96 / Phase error: 24.2741
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2183 1556 5 %
Rwork0.1754 29561 -
obs0.1775 31117 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 49.25 Å2
Refinement stepCycle: LAST / Resolution: 2.45→45.11 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5808 0 0 250 6058
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00495956
X-RAY DIFFRACTIONf_angle_d0.78488084
X-RAY DIFFRACTIONf_chiral_restr0.055854
X-RAY DIFFRACTIONf_plane_restr0.01711052
X-RAY DIFFRACTIONf_dihedral_angle_d13.57552172
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.45-2.530.36391390.30442639X-RAY DIFFRACTION99
2.53-2.620.3171430.27322706X-RAY DIFFRACTION100
2.62-2.720.32081420.24662704X-RAY DIFFRACTION100
2.72-2.850.27941420.23392688X-RAY DIFFRACTION100
2.85-30.29051400.22082676X-RAY DIFFRACTION100
3-3.190.26921420.20352700X-RAY DIFFRACTION100
3.19-3.430.23151420.18742686X-RAY DIFFRACTION100
3.43-3.780.23031420.16092699X-RAY DIFFRACTION99.96
3.78-4.320.17351420.14052693X-RAY DIFFRACTION100
4.32-5.440.16711400.1332673X-RAY DIFFRACTION100
5.45-45.110.16581420.14772697X-RAY DIFFRACTION99.82

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