scholarly journals Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

ACS Catalysis ◽  
2020 ◽  
Vol 10 (19) ◽  
pp. 11253-11267
Author(s):  
Anil R. Mhashal ◽  
Adrian Romero-Rivera ◽  
Lisa S. Mydy ◽  
Judith R. Cristobal ◽  
Andrew M. Gulick ◽  
...  
Keyword(s):  
Active Site ◽  
Hydride Transfer ◽  
Side Chains ◽  
Flexible Loop ◽  
Glycerol 3 Phosphate Dehydrogenase

scholarly journals Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

scholarly journals Localising individual atoms of tryptophan side chains in the metallo-<i>β</i>-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites

2022 ◽  
Vol 3 (1) ◽  
pp. 1-13
Author(s):  
Henry W. Orton ◽  
Iresha D. Herath ◽  
Ansis Maleckis ◽  
Shereen Jabar ◽  
Monika Szabo ◽  
...  
Keyword(s):  
Active Site ◽  
Side Chains ◽  
Pseudocontact Shifts ◽  
Magnetic Susceptibility Anisotropy ◽  
Single Crystal Structures ◽  
Flexible Loop ◽  
Backbone Amide ◽  
Tryptophan Residues ◽  
Atomic Coordinates ◽  
Tryptophan Side Chains

Abstract. The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

scholarly journals Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

2020 ◽  
Author(s):  
Anil Ranu Mhashal ◽  
Adrian Romero-Rivera ◽  
Lisa S. Mydy ◽  
Judith R. Cristobal ◽  
Andrew M. Gulick ◽  
...  
Keyword(s):  
Conformational Change ◽  
Critical Role ◽  
Valence Bond ◽  
Hydride Transfer ◽  
Structural Data ◽  
Wild Type ◽  
Human Form ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Important Enzyme

<div> <div> <div> <p>Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition state stabilization. While the human form (hlGPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend the experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hlGPDH and its variants, as well as providing a novel crystal structure of the binary hlGPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted variants of hlGPDH and investigated the effect of mutations on the catalysis from a detailed structural study. Our structural data and simulations also illustrate the critical role of the R269 side chain in facilitating the closure of hlGPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297), thus rationalizing a tremendous 41,000-fold decrease experimentally in the turnover number, kcat, upon truncating this residue. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of novel allosteric inhibitors targeting this biomedically important enzyme.</p></div></div></div>

scholarly journals Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

Studies on a Tyr residue critical for the binding of coenzyme and substrate in mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21): structure of the Y224D mutant enzyme

2010 ◽  
Vol 66 (2) ◽  
pp. 198-204
Author(s):  
Urmi Dhagat ◽  
Satoshi Endo ◽  
Hiroaki Mamiya ◽  
Akira Hara ◽  
Ossama El-Kabbani
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Unique Feature ◽  
Hydroxysteroid Dehydrogenase ◽  
Kinetic Constants ◽  
Mutant Enzyme ◽  
Side Chains ◽  
Wild Type ◽  
Stereospecific Reduction ◽  
Flexible Loop

Mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo–keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17)α-hydroxysteroids. The Y224D mutation of AKR1C21 reduced theKmvalue for NADP(H) by up to 80-fold and completely reversed the 17α stereospecificity of the enzyme. The crystal structure of the Y224D mutant at 2.3 Å resolution revealed that the mutation resulted in a change in the conformation of the flexible loop B, including the V-shaped groove, which is a unique feature of the active-site architecture of wild-type AKR1C21 and is formed by the side chains of Tyr224 and Trp227. Furthermore, mutations (Y224F and Q222N) of residues involved in forming the safety belt for binding of the coenzyme showed similar alterations in kinetic constants for 3α-hydroxy/3-ketosteroids and 17-hydroxy/ketosteroids compared with the wild type.

scholarly journals The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop

1997 ◽  
Vol 6 (2) ◽  
pp. 331-339 ◽  
Author(s):  
Michael Forstner ◽  
Alexandre Müller ◽  
Martin Stolz ◽  
Theo Wallimann
Keyword(s):  
Creatine Kinase ◽  
Active Site ◽  
Critical Role ◽  
Flexible Loop ◽  
Kinase A

scholarly journals Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

2020 ◽  
Author(s):  
Anil Ranu Mhashal ◽  
Adrian Romero-Rivera ◽  
Lisa S. Mydy ◽  
Judith R. Cristobal ◽  
Andrew M. Gulick ◽  
...  
Keyword(s):  
Conformational Change ◽  
Critical Role ◽  
Valence Bond ◽  
Hydride Transfer ◽  
Structural Data ◽  
Wild Type ◽  
Human Form ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Important Enzyme

<div> <div> <div> <p>Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition state stabilization. While the human form (hlGPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend the experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hlGPDH and its variants, as well as providing a novel crystal structure of the binary hlGPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted variants of hlGPDH and investigated the effect of mutations on the catalysis from a detailed structural study. Our structural data and simulations also illustrate the critical role of the R269 side chain in facilitating the closure of hlGPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297), thus rationalizing a tremendous 41,000-fold decrease experimentally in the turnover number, kcat, upon truncating this residue. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of novel allosteric inhibitors targeting this biomedically important enzyme.</p></div></div></div>

scholarly journals Localising individual atoms of tryptophan side chains in the metallo-β-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites

2021 ◽  
Author(s):  
Henry W. Orton ◽  
Iresha D. Herath ◽  
Ansis Maleckis ◽  
Shereen Jabar ◽  
Monika Szabo ◽  
...  
Keyword(s):  
Active Site ◽  
Side Chains ◽  
Pseudocontact Shifts ◽  
Magnetic Susceptibility Anisotropy ◽  
Single Crystal Structures ◽  
Flexible Loop ◽  
Backbone Amide ◽  
Tryptophan Residues ◽  
Atomic Coordinates ◽  
Tryptophan Side Chains

Abstract. The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCS) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is located in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

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