Conformational Changes During the Catalytic Cycle of Gluconate Kinase as Revealed by X-ray Crystallography

Numerous enzymes, such as the pyridoxal 5′-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

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The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

10.1101/389460 ◽

2018 ◽

Author(s):

Mohammed Jamshad ◽

Timothy J. Knowles ◽

Scott A. White ◽

Douglas G. Ward ◽

Fiyaz Mohammed ◽

...

Keyword(s):

Conformational Changes ◽

Metal Binding ◽

Cytoplasmic Membrane ◽

Protein Recognition ◽

X Ray ◽

Ribosome Binding ◽

X Ray Crystallography ◽

Substrate Protein ◽

Metal Binding Domain

AbstractIn bacteria, the translocation of a subset of proteins across the cytoplasmic membrane by the Sec machinery requires SecA. Although SecA can recognise nascent polypeptides, the mechanism of cotranslational substrate protein recognition is not known. Here, we investigated the role of the C-terminal tail (CTT) of SecA, which consists of a flexible linker (FLD) and a small metal-binding domain (MBD), in its interaction with nascent polypeptides. Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the entire CTT or the MBD alone had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function. Autophotocrosslinking, mass spectrometry, x-ray crystallography and small-angle x-ray scattering experiments provided insight into the CTT-mediated conformational changes in SecA. Finally, photocrosslinking experiments indicated that binding of SecA to substrate protein affected its interaction with the ribosome. Taken together, our results suggest a mechanism for substrate protein recognition.Impact StatementSecA is an evolutionarily conserved ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. We investigated how SecA recognises its substrate proteins at the ribosome as they are still being synthesised (i.e. cotranslationally).

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Structural basis of antagonizing the vitamin K catalytic cycle for anticoagulation

Science ◽

10.1126/science.abc5667 ◽

2020 ◽

Vol 371 (6524) ◽

pp. eabc5667 ◽

Cited By ~ 1

Author(s):

Shixuan Liu ◽

Shuang Li ◽

Guomin Shen ◽

Narayanasami Sukumar ◽

Andrzej M. Krezel ◽

...

Keyword(s):

Hydrogen Bonding ◽

Vitamin K ◽

Conformational Changes ◽

Vitamin K Antagonists ◽

Catalytic Cycle ◽

Structural Basis ◽

X Ray ◽

Redox States ◽

Hydrogen Bonding Interactions ◽

State Form

Vitamin K antagonists are widely used anticoagulants that target vitamin K epoxide reductases (VKOR), a family of integral membrane enzymes. To elucidate their catalytic cycle and inhibitory mechanism, we report 11 x-ray crystal structures of human VKOR and pufferfish VKOR-like, with substrates and antagonists in different redox states. Substrates entering the active site in a partially oxidized state form cysteine adducts that induce an open-to-closed conformational change, triggering reduction. Binding and catalysis are facilitated by hydrogen-bonding interactions in a hydrophobic pocket. The antagonists bind specifically to the same hydrogen-bonding residues and induce a similar closed conformation. Thus, vitamin K antagonists act through mimicking the key interactions and conformational changes required for the VKOR catalytic cycle.

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Interpenetrated Double Pillared-Layer CoII MOFs with pcu Topology

Australian Journal of Chemistry ◽

10.1071/ch16438 ◽

2017 ◽

Vol 70 (5) ◽

pp. 461 ◽

Cited By ~ 3

Author(s):

In-Hyeok Park ◽

Yunji Kang ◽

Eunji Lee ◽

Anjana Chanthapally ◽

Shim Sung Lee ◽

...

Keyword(s):

Conformational Changes ◽

Layer Structure ◽

Metal Organic Frameworks ◽

Trans Conformation ◽

X Ray ◽

Pore Sizes ◽

X Ray Crystallography ◽

Metal Organic

Three double pillared-layer CoII metal–organic frameworks (MOFs) with a pcu topology of a long, conformationally flexible, dipyridyl spacer ligand, 1,4-bis[2-(4-pyridyl)ethenyl]benzene (bpeb), and aromatic dicarboxylates (1,4-benzenedicarboxylate (bdc), 2,6-naphthalenedicarboxylate (ndc), and biphenyl-4,4′-dicarboxylate (bpdc)) have been synthesised and structurally characterised by X-ray crystallography. The MOFs are denoted as [Co2(bpeb)2(bdc)2]·DMF·3H2O (1), [Co2(bpeb)2(ndc)2]·1.75DMF·3.75H2O (2), and [Co2(bpeb)2(bpdc)2]·3.5DMF·4H2O (3). In the dinuclear repeating unit, four carboxylates are bonded to two CoII atoms forming a (4,4) layer structure. The axial positions are occupied by bpeb ligands. Of these, 1 and 2 have 2-fold interpenetration, whereas 3 displays 3-fold interpenetration. The two bpeb space ligands in 1 have trans,trans,trans and trans,cis,trans conformations. In contrast, the bpeb ligands in 2 and 3 have a trans,cis,trans conformation. Although the olefin groups in two adjacent bpeb ligands, as the double pillars in 2 and 3, satisfy the conditions for photo-dimerisation to occur, they are photo-inactive. The conformational changes of bpeb, bonding modes of the dicarboxylates, and pore sizes in these double pillared-layer compounds have been discussed.

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Ligand-induced Conformational Changes and a Reaction Intermediate in Branched-chain 2-Oxo Acid Dehydrogenase (E1) from Thermus thermophilus HB8, as Revealed by X-ray Crystallography

Journal of Molecular Biology ◽

10.1016/j.jmb.2004.02.011 ◽

2004 ◽

Vol 337 (4) ◽

pp. 1011-1033 ◽

Cited By ~ 26

Author(s):

Tadashi Nakai ◽

Noriko Nakagawa ◽

Nobuko Maoka ◽

Ryoji Masui ◽

Seiki Kuramitsu ◽

...

Keyword(s):

Conformational Changes ◽

Thermus Thermophilus ◽

Thermus Thermophilus Hb8 ◽

X Ray ◽

X Ray Crystallography ◽

Acid Dehydrogenase ◽

Reaction Intermediate ◽

Branched Chain

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Bambusuril analogs based on alternating glycoluril and xylylene units

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.15.124 ◽

2019 ◽

Vol 15 ◽

pp. 1268-1274 ◽

Cited By ~ 1

Author(s):

Tomáš Lízal ◽

Vladimír Šindelář

Keyword(s):

Nmr Spectroscopy ◽

Supramolecular Chemistry ◽

Conformational Changes ◽

Building Block ◽

Main Product ◽

Variable Temperature ◽

Guest Molecules ◽

X Ray ◽

X Ray Crystallography ◽

Design And Synthesis

The glycoluril monomer is a popular building block in supramolecular chemistry as it is used for the synthesis of versatile host molecules which can interact with cationic, anionic or neutral guest molecules. Here we present the design and synthesis of a new hybrid macrocycle containing glycoluril and aromatic units. The reaction afforded a mixture of macrocyclic homologues from which a two-membered macrocycle was isolated as the main product. Two disastereomers of the macrocycle were separated and characterized by means of NMR spectroscopy and X-ray crystallography. Conformational changes of these diastereomers were investigated using DFT models and variable-temperature NMR.

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Nanosecond pump–probe device for time-resolved serial femtosecond crystallography developed at SACLA

Journal of Synchrotron Radiation ◽

10.1107/s160057751701030x ◽

2017 ◽

Vol 24 (5) ◽

pp. 1086-1091 ◽

Cited By ~ 16

Author(s):

Minoru Kubo ◽

Eriko Nango ◽

Kensuke Tono ◽

Tetsunari Kimura ◽

Shigeki Owada ◽

...

Keyword(s):

Conformational Changes ◽

Free Electron ◽

Good Choice ◽

Cubic Phase ◽

Liquid Droplets ◽

Optical Pump ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography ◽

Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

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