6gi7: Difference between revisions
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<StructureSection load='6gi7' size='340' side='right'caption='[[6gi7]], [[Resolution|resolution]] 1.30Å' scene=''> | <StructureSection load='6gi7' size='340' side='right'caption='[[6gi7]], [[Resolution|resolution]] 1.30Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>[[6gi7]] is a 1 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6GI7 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6GI7 FirstGlance]. <br> | <table><tr><td colspan='2'>[[6gi7]] is a 1 chain structure with sequence from [http://en.wikipedia.org/wiki/"aerobacter_cloacae"_(jordan_1890)_bergey_et_al._1923 "aerobacter cloacae" (jordan 1890) bergey et al. 1923]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6GI7 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6GI7 FirstGlance]. <br> | ||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene>, <scene name='pdbligand=FMN:FLAVIN+MONONUCLEOTIDE'>FMN</scene></td></tr> | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene>, <scene name='pdbligand=FMN:FLAVIN+MONONUCLEOTIDE'>FMN</scene></td></tr> | ||
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">onr ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=550 "Aerobacter cloacae" (Jordan 1890) Bergey et al. 1923])</td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6gi7 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6gi7 OCA], [http://pdbe.org/6gi7 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6gi7 RCSB], [http://www.ebi.ac.uk/pdbsum/6gi7 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6gi7 ProSAT]</span></td></tr> | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6gi7 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6gi7 OCA], [http://pdbe.org/6gi7 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6gi7 RCSB], [http://www.ebi.ac.uk/pdbsum/6gi7 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6gi7 ProSAT]</span></td></tr> | ||
</table> | </table> | ||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR-coenzyme complexes. We find that mutagenesis of these second sphere "noncatalytic" residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR-NADH and PETNR-NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy-enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer. | |||
Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes.,Iorgu AI, Baxter NJ, Cliff MJ, Levy C, Waltho JP, Hay S, Scrutton NS ACS Catal. 2018 Dec 7;8(12):11589-11599. doi: 10.1021/acscatal.8b02810. Epub 2018, Oct 26. PMID:31119061<ref>PMID:31119061</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 6gi7" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
Latest revision as of 02:02, 6 June 2019
Crystal structure of pentaerythritol tetranitrate reductase (PETNR) mutant L25ICrystal structure of pentaerythritol tetranitrate reductase (PETNR) mutant L25I
Structural highlights
Publication Abstract from PubMedMany enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR-coenzyme complexes. We find that mutagenesis of these second sphere "noncatalytic" residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR-NADH and PETNR-NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy-enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer. Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes.,Iorgu AI, Baxter NJ, Cliff MJ, Levy C, Waltho JP, Hay S, Scrutton NS ACS Catal. 2018 Dec 7;8(12):11589-11599. doi: 10.1021/acscatal.8b02810. Epub 2018, Oct 26. PMID:31119061[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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