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- PDB-9j25: Structural basis of the bifunctionality of M. salinexigens ZYF650... -

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Basic information

Entry
Database: PDB / ID: 9j25
TitleStructural basis of the bifunctionality of M. salinexigens ZYF650T glucosylglycerol phosphorylase in glucosylglycerol catabolism
ComponentsSucrose phosphorylase
KeywordsSTRUCTURAL PROTEIN / Glucosylglycerol / phosphorylase-GH13_18 / structure-double displacement mechanism
Function / homology
Function and homology information


sucrose phosphorylase / sucrose phosphorylase activity / carbohydrate metabolic process
Similarity search - Function
Sucrose phosphorylase / Oligo-1,6-glucosidase, domain 2 / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycoside hydrolase superfamily
Similarity search - Domain/homology
DI(HYDROXYETHYL)ETHER / PHOSPHATE ION / Sucrose phosphorylase
Similarity search - Component
Biological speciesMarinobacter salinexigens (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.74 Å
AuthorsLu, D. / Ma, H.L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32271358 China
CitationJournal: J Biol Chem / Year: 2025
Title: Structural basis of the bifunctionality of Marinobacter salinexigens ZYF650 glucosylglycerol phosphorylase in glucosylglycerol catabolism.
Authors: Di Lu / Keke Zhang / Chen Cheng / Danni Wu / Lei Yin / Quan Luo / Meiyun Shi / Honglei Ma / Xuefeng Lu /
Abstract: 2-O-α-Glucosylglycerol (GG) is a natural heteroside synthesized by many cyanobacteria and a few heterotrophic bacteria under salt stress conditions. Bacteria produce GG in response to stimuli and ...2-O-α-Glucosylglycerol (GG) is a natural heteroside synthesized by many cyanobacteria and a few heterotrophic bacteria under salt stress conditions. Bacteria produce GG in response to stimuli and degrade it once the stimulus diminishes. Heterotrophic bacteria utilize GG phosphorylase (GGP), a member of the GH13_18 family, via a two-step process consisting of phosphorolysis and hydrolysis for GG catabolism. However, the precise mechanism by which GGP degrades GG remains elusive. We determined the 3D structure of a recently identified GGP (MsGGP) of the deep-sea bacterium Marinobacter salinexigens ZYF650, in complex with glucose and glycerol, α-d-glucose-1-phosphate (αGlc1-P), and orthophosphate (inorganic phosphate) at resolutions of 2.5, 2.7, and 2.7 Å, respectively. Notably, the first αGlc1-P complex structure in the GH13_18 family, the complex of MsGGP and αGlc1-P, validates that GGP catalyzes GG decomposition through consecutive phosphorolysis and hydrolysis. In addition, the structure reveals the mechanism of high stereoselectivity on αGlc1-P. Glu231 and Asp190 were identified as the catalytic residues. Interestingly, these structures closely resemble each other, indicating minimal conformational changes upon binding end-product glucose and glycerol, or the intermediate αGlc1-P. The structures also indicate that the substrates may follow a specific trajectory and a precise order toward the active center in close proximity and in a geometrically favorable orientation for catalysis in a double displacement mechanism.
History
DepositionAug 6, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sucrose phosphorylase
B: Sucrose phosphorylase
C: Sucrose phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,41710
Polymers164,7123
Non-polymers7057
Water2,918162
1
A: Sucrose phosphorylase
B: Sucrose phosphorylase
C: Sucrose phosphorylase
hetero molecules

A: Sucrose phosphorylase
B: Sucrose phosphorylase
C: Sucrose phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)330,83420
Polymers329,4236
Non-polymers1,41114
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_554-x,y,-z-1/21
Buried area16140 Å2
ΔGint-103 kcal/mol
Surface area95510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)107.244, 176.117, 177.369
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11A-603-

HOH

21B-641-

HOH

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Components

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Protein , 1 types, 3 molecules ABC

#1: Protein Sucrose phosphorylase / Glucosylglycerol phosphorylase


Mass: 54903.852 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Marinobacter salinexigens (bacteria) / Gene: gtfA, FWJ25_14990 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5B0VBK8, sucrose phosphorylase

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Non-polymers , 5 types, 169 molecules

#2: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#3: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#4: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 162 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
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: 2.54 Å3/Da / Density % sol: 51.62 %
Crystal growTemperature: 289 K / Method: vapor diffusion
Details: 1.8 M Ammonium sulfate, 0.1 M BIS-TRIS pH 6.5, 2% Polyethylene glycol monomethyl ether 550

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL18U1 / Wavelength: 0.987 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 17, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 2.74→50 Å / Num. obs: 44128 / % possible obs: 90.47 % / Redundancy: 12.3 % / R split: 0.055 / Rrim(I) all: 0.196 / Net I/σ(I): 8.4
Reflection shellResolution: 2.75→2.8 Å / Num. unique obs: 2165 / Rpim(I) all: 0.348

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Processing

Software
NameVersionClassification
PHENIX(1.17.1_3660: ???)refinement
HKL-30007.21data reduction
HKL-30007.21data scaling
PHASER2.7.0phasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7XDQ

7xdq


Resolution: 2.74→49.09 Å / SU ML: 0.38 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 28.09 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2597 1911 4.78 %
Rwork0.2222 --
obs0.224 40013 90.47 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.74→49.09 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10622 0 30 162 10814
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00710925
X-RAY DIFFRACTIONf_angle_d1.05714845
X-RAY DIFFRACTIONf_dihedral_angle_d16.1971443
X-RAY DIFFRACTIONf_chiral_restr0.0571612
X-RAY DIFFRACTIONf_plane_restr0.0071923
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.74-2.810.3152780.25891620X-RAY DIFFRACTION54
2.81-2.890.34181130.2731951X-RAY DIFFRACTION66
2.89-2.970.34221290.27172312X-RAY DIFFRACTION78
2.97-3.070.29311050.27752595X-RAY DIFFRACTION87
3.07-3.180.33721630.27392793X-RAY DIFFRACTION95
3.18-3.310.32581380.2612969X-RAY DIFFRACTION99
3.31-3.460.29031300.25483003X-RAY DIFFRACTION99
3.46-3.640.28451370.26792899X-RAY DIFFRACTION97
3.64-3.870.37451240.31122904X-RAY DIFFRACTION96
3.87-4.170.25821840.22952864X-RAY DIFFRACTION96
4.17-4.590.19581660.16142981X-RAY DIFFRACTION100
4.59-5.250.2051540.15783033X-RAY DIFFRACTION100
5.25-6.610.23281580.18153057X-RAY DIFFRACTION100
6.61-49.090.18541320.17913121X-RAY DIFFRACTION98
Refinement TLS params.

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

IDL11 (°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)
11.07260.13690.03130.30810.26351.4288-0.0602-0.21510.09580.0681-0.06210.0781-0.3953-0.30540.05970.39610.1199-0.04840.2525-0.02360.3202-9.0716-2.1645-22.006
21.8043-0.36390.29351.89360.52272.469-0.143-0.1264-0.05790.07130.10060.0941-0.08850.14160.05750.3372-0.031-0.04330.20230.01420.318115.6945-7.2129-29.0638
31.22360.5738-0.56511.2548-0.62471.69140.1914-0.10820.24420.1311-0.1759-0.22020.16320.69060.06440.25340.2176-0.07330.6864-0.05650.431438.8445-49.8963-20.3418
40.85510.10260.04561.2018-0.22230.78530.0351-0.02520.25530.1453-0.1838-0.1998-0.09170.6640.08780.18920.085-0.05890.7524-0.01650.385539.5185-35.2257-34.7743
50.99940.3540.17181.2783-1.15361.33920.2745-0.25120.2960.121-0.24560.1719-0.3956-0.0176-0.05520.3830.06920.00160.4611-0.06120.39516.987-47.8811-35.0893
61.16410.19650.6361.3322-0.30980.47810.38660.1541-0.1608-0.1116-0.14290.05810.79710.18530.01430.61180.1841-0.10160.3066-0.00270.284522.3432-64.3243-29.607
71.2618-0.75310.58480.95170.15422.74070.1187-0.0834-0.05110.7862-0.21210.06930.773-0.1454-0.14080.7508-0.2546-0.04270.5527-0.02610.31-26.2651-51.0895-6.0762
80.6976-0.4070.92544.0064-1.16181.3603-0.0179-0.2539-0.01231.0777-0.0233-1.18540.6872-0.03110.0240.8568-0.0953-0.1660.4540.06140.5817-18.7455-59.8553-8.1036
90.6170.0546-0.17191.1142-0.2240.5690.0829-0.1107-0.19430.2715-0.10150.04630.7089-0.2042-0.03730.7639-0.3519-0.11650.35190.08370.3202-15.0669-64.4651-23.6336
102.74371.8426-1.68463.6505-1.38141.2579-0.2135-0.4282-0.0081-0.0878-0.2834-0.39720.36380.3845-0.1860.3529-0.1034-0.03670.3926-0.07480.4013-14.9707-43.8281-29.2674
110.55380.0626-0.14592.6750.21151.99480.0725-0.02740.1320.40510.06510.0363-0.0032-0.5836-0.06250.2646-0.08250.02770.4354-0.01550.285-29.0516-36.127-18.0291
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 276 )
2X-RAY DIFFRACTION2chain 'A' and (resid 277 through 480 )
3X-RAY DIFFRACTION3chain 'B' and (resid 1 through 66 )
4X-RAY DIFFRACTION4chain 'B' and (resid 67 through 264 )
5X-RAY DIFFRACTION5chain 'B' and (resid 265 through 348 )
6X-RAY DIFFRACTION6chain 'B' and (resid 349 through 480 )
7X-RAY DIFFRACTION7chain 'C' and (resid 1 through 30 )
8X-RAY DIFFRACTION8chain 'C' and (resid 31 through 65 )
9X-RAY DIFFRACTION9chain 'C' and (resid 66 through 276 )
10X-RAY DIFFRACTION10chain 'C' and (resid 277 through 311 )
11X-RAY DIFFRACTION11chain 'C' and (resid 312 through 480 )

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