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- PDB-8j5d: Cryo-EM structure of starch degradation complex of BAM1-LSF1-MDH -

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Basic information

Entry
Database: PDB / ID: 8j5d
TitleCryo-EM structure of starch degradation complex of BAM1-LSF1-MDH
Components
  • Beta-amylase 1, chloroplastic
  • Malate dehydrogenase, chloroplastic
  • Phosphoglucan phosphatase LSF1, chloroplastic
KeywordsHYDROLASE / amylase / starch degradation
Function / homology
Function and homology information


chloroplast starch grain / chloroplast protein-transporting ATPase activity / starch grain / Ycf2/FtsHi complex / plastid stroma / carbohydrate phosphatase activity / protein import into chloroplast stroma / : / chloroplast inner membrane / stromule ...chloroplast starch grain / chloroplast protein-transporting ATPase activity / starch grain / Ycf2/FtsHi complex / plastid stroma / carbohydrate phosphatase activity / protein import into chloroplast stroma / : / chloroplast inner membrane / stromule / chloroplast organization / beta-amylase / beta-amylase activity / amylopectin maltohydrolase activity / starch catabolic process / protein tyrosine/serine/threonine phosphatase activity / malate dehydrogenase / embryo development ending in seed dormancy / L-malate dehydrogenase (NAD+) activity / response to water deprivation / malate metabolic process / Hydrolases; Acting on ester bonds; Phosphoric-monoester hydrolases / plant-type vacuole / apoplast / chloroplast envelope / plastid / chloroplast stroma / dephosphorylation / tricarboxylic acid cycle / response to cold / chloroplast / defense response to bacterium / mitochondrion / cytosol
Similarity search - Function
Laforin-like, dual specificity phosphatase domain / Glycoside hydrolase, family 14B, plant / Malate dehydrogenase, type 1 / Malate dehydrogenase, active site / Malate dehydrogenase active site signature. / Beta-amylase active site 2. / Glycoside hydrolase, family 14, conserved site / Beta-amylase active site 1. / Glycoside hydrolase, family 14 / Glycosyl hydrolase family 14 ...Laforin-like, dual specificity phosphatase domain / Glycoside hydrolase, family 14B, plant / Malate dehydrogenase, type 1 / Malate dehydrogenase, active site / Malate dehydrogenase active site signature. / Beta-amylase active site 2. / Glycoside hydrolase, family 14, conserved site / Beta-amylase active site 1. / Glycoside hydrolase, family 14 / Glycosyl hydrolase family 14 / AMP-activated protein kinase, glycogen-binding domain / Glycogen recognition site of AMP-activated protein kinase / Dual specificity phosphatase, catalytic domain / Dual specificity phosphatase, catalytic domain / Dual specificity phosphatase, catalytic domain / Dual specificity protein phosphatase domain / Dual specificity protein phosphatase domain profile. / Lactate/malate dehydrogenase, N-terminal / Lactate/malate dehydrogenase, C-terminal / lactate/malate dehydrogenase, NAD binding domain / lactate/malate dehydrogenase, alpha/beta C-terminal domain / Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal / Protein-tyrosine phosphatase-like / PDZ superfamily / Immunoglobulin E-set / Glycoside hydrolase superfamily / NAD(P)-binding domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Phosphoglucan phosphatase LSF1, chloroplastic / Beta-amylase 1, chloroplastic / Malate dehydrogenase, chloroplastic
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsGuan, Z.Y. / Liu, J. / Yan, J.J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Plant Cell / Year: 2023
Title: The LIKE SEX FOUR 1-malate dehydrogenase complex functions as a scaffold to recruit β-amylase to promote starch degradation.
Authors: Jian Liu / Xuecui Wang / Zeyuan Guan / Menglong Wu / Xinyue Wang / Rong Fan / Fei Zhang / Junjun Yan / Yanjun Liu / Delin Zhang / Ping Yin / Junjie Yan /
Abstract: In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to ...In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including β-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit β-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex. The starch hydrolysis activity of BAM1 drastically increased in the presence of LSF1-MDH in vitro. We determined the structure of the BAM1-LSF1-MDH complex by a combination of cryo-electron microscopy, crosslinking mass spectrometry, and molecular docking. The starch-binding domain of the dual-specificity phosphatase and carbohydrate-binding module of LSF1 was docked in proximity to BAM1, thus facilitating BAM1 access to and hydrolysis of the polyglucans of starch, thus revealing the molecular mechanism by which the LSF1-MDH complex improves the starch degradation activity of BAM1. Moreover, LSF1 is phosphatase inactive, and the enzymatic activity of MDH was dispensable for starch degradation, suggesting nonenzymatic scaffold functions for LSF1-MDH in starch degradation. These findings provide important insights into the precise regulation of starch degradation.
History
DepositionApr 21, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Beta-amylase 1, chloroplastic
B: Malate dehydrogenase, chloroplastic
D: Phosphoglucan phosphatase LSF1, chloroplastic
C: Malate dehydrogenase, chloroplastic


Theoretical massNumber of molelcules
Total (without water)183,5834
Polymers183,5834
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Beta-amylase 1, chloroplastic / BAM1 / 1 / 4-alpha-D-glucan maltohydrolase / Beta-amylase 7 / Thioredoxin-regulated beta-amylase / TR-BAMY


Mass: 56111.992 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: BAM1, BMY7, TRBAMY, At3g23920, F14O13.12 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9LIR6, beta-amylase
#2: Protein Malate dehydrogenase, chloroplastic / Chloroplastic malate dehydrogenase / Chloroplastic MDH / cpNAD-MDH / Plastidic NAD-dependent malate ...Chloroplastic malate dehydrogenase / Chloroplastic MDH / cpNAD-MDH / Plastidic NAD-dependent malate dehydrogenase / pNAD-MDH


Mass: 34120.137 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: At3g47520, F1P2.70 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9SN86, malate dehydrogenase
#3: Protein Phosphoglucan phosphatase LSF1, chloroplastic / Phosphoglucan phosphatase like sex Four1 / Protein LIKE SEX4 1


Mass: 59231.191 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: LSF1, PTPKIS2, At3g01510, F4P13.6 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: F4J117, Hydrolases; Acting on ester bonds; Phosphoric-monoester hydrolases

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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: BAM1-LSF1-MDH complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: cryoSPARC / Category: classification
CTF correctionType: NONE
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 305907 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0079727
ELECTRON MICROSCOPYf_angle_d0.74713191
ELECTRON MICROSCOPYf_dihedral_angle_d4.3021315
ELECTRON MICROSCOPYf_chiral_restr0.0441501
ELECTRON MICROSCOPYf_plane_restr0.0041699

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