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- PDB-6jcv: Cryo-EM structure of Sulfolobus solfataricus ketol-acid reductois... -

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Entry
Database: PDB / ID: 6jcv
TitleCryo-EM structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) with Mg2+ at pH7.5
ComponentsPutative ketol-acid reductoisomerase 2
KeywordsISOMERASE / Bi-specific / Thermostable / Reductoisomerase / Magnesium-dependent / Dodecamer / Knot-structure
Function / homology
Function and homology information


ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / L-valine biosynthetic process / isoleucine biosynthetic process
Similarity search - Function
Ketol-acid reductoisomerase, C-terminal / Ketol-acid reductoisomerase / Ketol-acid reductoisomerase, N-terminal / Acetohydroxy acid isomeroreductase, catalytic domain / Acetohydroxy acid isomeroreductase, NADPH-binding domain / KARI N-terminal domain profile. / KARI C-terminal domain profile. / 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily ...Ketol-acid reductoisomerase, C-terminal / Ketol-acid reductoisomerase / Ketol-acid reductoisomerase, N-terminal / Acetohydroxy acid isomeroreductase, catalytic domain / Acetohydroxy acid isomeroreductase, NADPH-binding domain / KARI N-terminal domain profile. / KARI C-terminal domain profile. / 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Putative ketol-acid reductoisomerase 2
Similarity search - Component
Biological speciesSaccharolobus solfataricus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsChen, C.Y. / Chang, Y.C. / Lin, K.F. / Huang, C.H. / Lin, B.L. / Ko, T.P. / Hsieh, D.L. / Tsai, M.D.
Funding support Taiwan, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (Taiwan)AS-KPQ-105-TPP Taiwan
Ministry of Science and Technology (Taiwan)OST 106-2113-M-008-011 Taiwan
CitationJournal: J Am Chem Soc / Year: 2019
Title: Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase.
Authors: Chin-Yu Chen / Yuan-Chih Chang / Bo-Lin Lin / Kuan-Fu Lin / Chun-Hsiang Huang / Dong-Lin Hsieh / Tzu-Ping Ko / Ming-Daw Tsai /
Abstract: While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 ...While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus (Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for Sso-KARI:2Mg. The results showed that the distances of the two catalytic Mg ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two Sso-KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking "induced-fit", reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology.
History
DepositionJan 30, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 17, 2019Provider: repository / Type: Initial release
Revision 1.1May 1, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Putative ketol-acid reductoisomerase 2
B: Putative ketol-acid reductoisomerase 2
C: Putative ketol-acid reductoisomerase 2
D: Putative ketol-acid reductoisomerase 2
E: Putative ketol-acid reductoisomerase 2
F: Putative ketol-acid reductoisomerase 2
G: Putative ketol-acid reductoisomerase 2
H: Putative ketol-acid reductoisomerase 2
I: Putative ketol-acid reductoisomerase 2
J: Putative ketol-acid reductoisomerase 2
K: Putative ketol-acid reductoisomerase 2
L: Putative ketol-acid reductoisomerase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)447,34236
Polymers446,75812
Non-polymers58324
Water1,72996
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Putative ketol-acid reductoisomerase 2 / Acetohydroxy-acid isomeroreductase 2 / Alpha-keto-beta-hydroxylacyl reductoisomerase 2


Mass: 37229.855 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) (archaea)
Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: ilvC2, ilvC-2, SSO1322 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: Q97YJ9, ketol-acid reductoisomerase (NADP+)
#2: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 96 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) Dodecamer in complex with Mg2+ in pH7.5
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 446 kDa/nm / Experimental value: YES
Source (natural)Organism: Sulfolobus solfataricus P2 (archaea)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl(HOCH2)3CNH21
250 mMSodium chlorideNaCl1
35 mMMagnesium chlorideMgCl21
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 1.91 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cisTEM1.0.0-betaparticle selection
4cisTEM1.0.0-betaCTF correction
7UCSF Chimera1.13.1model fitting
8Coot0.8.9.1model fitting
10PHENIXdev-3339model refinement
11cisTEM1.0.0-betainitial Euler assignment
12cisTEM1.0.0-betafinal Euler assignment
13cisTEM1.0.0-betaclassification
14cisTEM1.0.0-beta3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34743 / Symmetry type: POINT
Atomic model buildingB value: 123.03 / Protocol: AB INITIO MODEL / Space: REAL

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