Details

Autor(en) / Beteiligte

• Nakata, Kunio
• Miyazaki, Naoyuki
• Yamaguchi, Hiroki
• Hirose, Mika
• Kashiwagi, Tatsuki
• Kutumbarao, Nidamarthi H.V.
• Miyashita, Osamu
• Tama, Florence
• Miyano, Hiroshi
• Mizukoshi, Toshimi
• Iwasaki, Kenji

Titel

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

Ist Teil von

• Journal of structural biology, 2022-06, Vol.214 (2), p.107842-107842, Article 107842

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2022

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• [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.