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

Nakata, Kengo; Miyazaki, Naoyuki; Yamaguchi, Hideyuki; Hirose, Mika; Kashiwagi, Tatsuki; Kutumbarao, N.H.V.; Miyashita, Osamu; Tama, Florence; Miyano, Hiroshi; Mizukoshi, Toshimi; Iwasaki, Katsunori

doi:10.1016/j.jsb.2022.107842

Cited by 5 publications

(7 citation statements)

References 37 publications

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“…2a). The recently solved cryo-EM structure of BlPKT (the BlPKT EM structure; PDB entry 6lxv; Nakata et al, 2022) also possessed the same octameric structure with D4 symmetry. The BbPKT crystal structures also possessed a similar octameric structure with I222 crystallographic symmetry (Suzuki et al, 2010).…”

Section: Overall Structures Of Blpkt Solved By the Xfel Sfx Methodsmentioning

confidence: 92%

“…Other factors, including the species of origin, the pH conditions during crystallization and the effects of crystal packing, do not seem to influence conformational changes in the QN-loop. In the recently solved BlPKT EM structure (PDB entry 6lxv; Nakata et al, 2022), the QN-loop adopts double conformations (conformers I and II). Conformer I coincides well with the 'open-form' conformation of the QN-loop in the BlPKT XFEL structure and conformer II is similar to the 'closed-form' conformation of the QN-loop in the BlPKT cryo structure (Fig.…”

Section: Active-site Structures Of Blpkt Solved By the Xfel Sfx Methodsmentioning

confidence: 99%

“…4d). Compared with the BlPKT EM structure, the BlPKT cryo structure may reflect the state of the target molecule in a more extreme environment with respect to temperature (Nakata et al, 2022). Although the reason why the QN-loops of PKT in cryogenic structures converge to the 'closed form' and those in ambient structures converge to the 'open form' needs to be further examined in future research, the 'closed form' and 'open form' of the QN-loop in the BlPKT holoenzyme may be the most stable conformations at cryogenic and ambient temperatures, respectively.…”

Section: Active-site Structures Of Blpkt Solved By the Xfel Sfx Methodsmentioning

confidence: 99%

“…The BlPKT protein solution was prepared according to the method described previously (Nakata et al, 2022). Equal amounts of 15 mg ml À1 BlPKT solution (4 mM DTT, 10 mg ml À1 TPP) and precipitant solution [50 mM malonate (Nacalai Tesque), 12% Tacsimate pH 5 (Hampton Research), 17% PEG 3350 (Hampton Research)] were mixed in order to prepare seed crystals.…”

Section: Preparation Of Blpkt Microcrystalsmentioning

confidence: 99%

“…Because of the high sequence identity between these two PKTs (84%), their three-dimensional structures are almost identical. Recently, the holoenzyme structure of BlPKT has been also determined by cryo-EM single-particle analysis (PDB entry 6lxv; Nakata et al, 2022). Although the structures of complexes of BbPKT with its reaction intermediates before and after dehydration [,-dihydroxyethyl TPP (DHETPP) and 2-acetyl-TPP (AcTPP)] together with its second substrate, P i , have been determined (PDB entries 3ahe, 3ahd and 3ahf, respectively; Suzuki et al, 2010), the structures of its complexes with the substrates X5P and F6P or with the inhibitor PEP are still unknown.…”

Section: Introductionmentioning

confidence: 99%

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Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography

Nakata

Kashiwagi

Kunishima

et al. 2023

Acta Cryst Sect D Struct Biol

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Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (Pi) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.5 Å resolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.

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Section: Overall Structures Of Blpkt Solved By the Xfel Sfx Methodsmentioning

confidence: 92%

Section: Active-site Structures Of Blpkt Solved By the Xfel Sfx Methodsmentioning

confidence: 99%

Section: Active-site Structures Of Blpkt Solved By the Xfel Sfx Methodsmentioning

confidence: 99%

Section: Preparation Of Blpkt Microcrystalsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography

Nakata

Kashiwagi

Kunishima

et al. 2023

Acta Cryst Sect D Struct Biol

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Frontiers in the enzymology of thiamin diphosphate-dependent enzymes

Prajapati

Pappenheim

Tittmann

2022

Current Opinion in Structural Biology

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An ATP-sensitive phosphoketolase regulates carbon fixation in cyanobacteria

Chang

Wang

et al. 2023

Nat Metab

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Regulation of CO2 fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP-sensing mechanism, where a drop in ATP level allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin–Benson–Bassham cycle. Deleting the SeXPK gene increased CO2 fixation particularly during light–dark transitions. In high-density cultures, the Δxpk strain showed a 60% increase in carbon fixation and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.

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

Cited by 5 publications

References 37 publications

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography

Frontiers in the enzymology of thiamin diphosphate-dependent enzymes

An ATP-sensitive phosphoketolase regulates carbon fixation in cyanobacteria

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