High-resolution structure of phosphoketolase from Bifidobacterium longum determined by cryo-EM single-particle analysis

[Display omitted] •3D structure of Bifidobacterium longum phosphoketolase was determined at 2.1 Å resolution by the cryo-EM single particle analysis.•Structural difference between cryo-EM and XRD structures: almost identical except for a small loop (QN-loop) near the substrate binding site.•QN-loop...

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Veröffentlicht in:Journal of structural biology 2022-06, Vol.214 (2), p.107842-107842, Article 107842
Hauptverfasser: Nakata, Kunio, Miyazaki, Naoyuki, Yamaguchi, Hiroki, Hirose, Mika, Kashiwagi, Tatsuki, Kutumbarao, Nidamarthi H.V., Miyashita, Osamu, Tama, Florence, Miyano, Hiroshi, Mizukoshi, Toshimi, Iwasaki, Kenji
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container_issue 2
container_start_page 107842
container_title Journal of structural biology
container_volume 214
creator Nakata, Kunio
Miyazaki, Naoyuki
Yamaguchi, Hiroki
Hirose, Mika
Kashiwagi, Tatsuki
Kutumbarao, Nidamarthi H.V.
Miyashita, Osamu
Tama, Florence
Miyano, Hiroshi
Mizukoshi, Toshimi
Iwasaki, Kenji
description [Display omitted] •3D structure of Bifidobacterium longum phosphoketolase was determined at 2.1 Å resolution by the cryo-EM single particle analysis.•Structural difference between cryo-EM and XRD structures: almost identical except for a small loop (QN-loop) near the substrate binding site.•QN-loop in the cryo-EM structure adopted multi-conformers (closed and open states) which were similar to the loop conformations in the substrate-free XRD structure of phosphoketolase and in the substrate-bound structure of transketolase, respectively.•It was revealed that essentially important coordinated water molecules can be detected in the cryo-EM structure as well as in the XRD structure. In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called “bifid shunt.” The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546–D547–H548–N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to “closed” and “open” states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.
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In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called “bifid shunt.” The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546–D547–H548–N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to “closed” and “open” states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. 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In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called “bifid shunt.” The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546–D547–H548–N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to “closed” and “open” states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.</description><subject>Aldehyde-Lyases - chemistry</subject><subject>Atomic resolution</subject><subject>Bifidobacterium longum - enzymology</subject><subject>Conformational change</subject><subject>Cryo-EM</subject><subject>Cryoelectron Microscopy - methods</subject><subject>Escherichia coli</subject><subject>Models, Molecular</subject><subject>Multi-conformer</subject><subject>Phosphoketolase</subject><subject>Single-particle analysis</subject><subject>Thiamine Pyrophosphate</subject><subject>Water</subject><issn>1047-8477</issn><issn>1095-8657</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kE9P3DAQxS1UVCj0A3CpfOSSxX9iJxGngmhBAnHhbjn2ePHWiRfbKdpvX6-W9tiRRjNPevOk-SF0QcmKEiqvNqtNHleMMFZ117fsCJ1SMoiml6L7tN_brunbrjtBX3LeEEJayuhndMIF7WkrulP0fu_Xr02CHMNSfJxxLmkxZUmAo8Pb15hr_4ISg86AXYoTvvHO2zhqUyD5ZcIhzus6LFQ9-RksHnfYpF1s7p5w9vM6QLPVqXgTAOtZh132-RwdOx0yfP2YZ-jlx93L7X3z-Pzz4fb7Y2NayUpjrXNCMts6QankIAchuBmGkfUDF3IwjgvXG0cHY9zILTBBdCe5qMUt5Wfo8hC7TfFtgVzU5LOBEPQMccmKSU4GJnrCqpUerCbFnBM4tU1-0mmnKFF73GqjKm61x60OuOvNt4_4ZZzA_rv4y7carg8GqD_-9pBUNh5mA9YnMEXZ6P8T_weSUZKN</recordid><startdate>202206</startdate><enddate>202206</enddate><creator>Nakata, Kunio</creator><creator>Miyazaki, Naoyuki</creator><creator>Yamaguchi, Hiroki</creator><creator>Hirose, Mika</creator><creator>Kashiwagi, Tatsuki</creator><creator>Kutumbarao, Nidamarthi H.V.</creator><creator>Miyashita, Osamu</creator><creator>Tama, Florence</creator><creator>Miyano, Hiroshi</creator><creator>Mizukoshi, Toshimi</creator><creator>Iwasaki, Kenji</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>202206</creationdate><title>High-resolution structure of phosphoketolase from Bifidobacterium longum determined by cryo-EM single-particle analysis</title><author>Nakata, Kunio ; 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In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called “bifid shunt.” The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546–D547–H548–N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to “closed” and “open” states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>35181457</pmid><doi>10.1016/j.jsb.2022.107842</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record>
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subjects Aldehyde-Lyases - chemistry
Atomic resolution
Bifidobacterium longum - enzymology
Conformational change
Cryo-EM
Cryoelectron Microscopy - methods
Escherichia coli
Models, Molecular
Multi-conformer
Phosphoketolase
Single-particle analysis
Thiamine Pyrophosphate
Water
title High-resolution structure of phosphoketolase from Bifidobacterium longum determined by cryo-EM single-particle analysis
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