6uqb: Difference between revisions
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==Crystal structure of R471A variant of cytosolic fumarate hydratase from Leishmania major in a complex with S-malate and malonate== | |||
<StructureSection load='6uqb' size='340' side='right'caption='[[6uqb]], [[Resolution|resolution]] 1.95Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[6uqb]] is a 2 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6UQB OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6UQB FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=GOL:GLYCEROL'>GOL</scene>, <scene name='pdbligand=LMR:(2S)-2-HYDROXYBUTANEDIOIC+ACID'>LMR</scene>, <scene name='pdbligand=MLA:MALONIC+ACID'>MLA</scene>, <scene name='pdbligand=SF4:IRON/SULFUR+CLUSTER'>SF4</scene></td></tr> | |||
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[6unz|6unz]], [[6uo0|6uo0]], [[6uoi|6uoi]], [[6uoj|6uoj]], [[6up9|6up9]], [[6upm|6upm]], [[6upo|6upo]], [[6uq8|6uq8]], [[6uq9|6uq9]], [[6uql|6uql]], [[6uqm|6uqm]], [[6uqn|6uqn]]</td></tr> | |||
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Fumarate_hydratase Fumarate hydratase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.2 4.2.1.2] </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=6uqb FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6uqb OCA], [http://pdbe.org/6uqb PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6uqb RCSB], [http://www.ebi.ac.uk/pdbsum/6uqb PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6uqb ProSAT]</span></td></tr> | |||
</table> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Class I fumarate hydratases (FHs) are central metabolic enzymes that use a [4Fe-4S] cluster to catalyze the reversible conversion of fumarate to S-malate. The parasite Leishmania major, which is responsible for leishmaniasis, expresses two class I FH isoforms: mitochondrial LmFH-1 and cytosolic LmFH-2. In this study, we present kinetic characterizations of both LmFH isoforms, present 13 crystal structures of LmFH-2 variants, and employ site-directed mutagenesis to investigate the enzyme's mechanism. Our kinetic data confirm that both LmFH-1 and LmFH-2 are susceptible to oxygen-dependent inhibition, with data from crystallography and electron paramagnetic resonance spectroscopy showing that oxygen exposure converts an active [4Fe-4S] cluster to an inactive [3Fe-4S] cluster. Our anaerobically conducted kinetic studies reveal a preference for fumarate over S-malate. Our data further reveal that single alanine substitutions of T467, R421, R471, D135, and H334 decrease kcat values 9-16000-fold without substantially affecting Km values, suggesting that these residues function in catalytic roles. Crystal structures of LmFH-2 variants are consistent with this idea, showing similar bidentate binding to the unique iron of the [4Fe-4S] cluster for substrate S-malate as observed in wild type FH. We further present LmFH-2 structures with substrate fumarate and weak inhibitors succinate and malonate bound in the active site and the first structure of an LmFH that is substrate-free and inhibitor-free, the latter showing increased mobility in the C-terminal domain. Collectively, these data provide insight into the molecular basis for the reaction catalyzed by LmFHs, enzymes that are potential drug targets against leishmaniasis. | |||
Structural and Biochemical Investigations of the [4Fe-4S] Cluster-Containing Fumarate Hydratase from Leishmania major.,Feliciano PR, Drennan CL Biochemistry. 2019 Nov 27. doi: 10.1021/acs.biochem.9b00923. PMID:31743022<ref>PMID:31743022</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
<div class="pdbe-citations 6uqb" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Fumarate hydratase]] | |||
[[Category: Large Structures]] | |||
[[Category: Drennan, C L]] | |||
[[Category: Feliciano, P R]] | |||
[[Category: Fumarase]] | |||
[[Category: Inhibitor]] | |||
[[Category: Leishmania major]] | |||
[[Category: Lyase]] | |||
[[Category: Malonate]] | |||
[[Category: S-malate]] | |||
[[Category: Substrate]] | |||
[[Category: Variant h334a]] | |||
Revision as of 11:26, 4 December 2019
Crystal structure of R471A variant of cytosolic fumarate hydratase from Leishmania major in a complex with S-malate and malonateCrystal structure of R471A variant of cytosolic fumarate hydratase from Leishmania major in a complex with S-malate and malonate
Structural highlights
Publication Abstract from PubMedClass I fumarate hydratases (FHs) are central metabolic enzymes that use a [4Fe-4S] cluster to catalyze the reversible conversion of fumarate to S-malate. The parasite Leishmania major, which is responsible for leishmaniasis, expresses two class I FH isoforms: mitochondrial LmFH-1 and cytosolic LmFH-2. In this study, we present kinetic characterizations of both LmFH isoforms, present 13 crystal structures of LmFH-2 variants, and employ site-directed mutagenesis to investigate the enzyme's mechanism. Our kinetic data confirm that both LmFH-1 and LmFH-2 are susceptible to oxygen-dependent inhibition, with data from crystallography and electron paramagnetic resonance spectroscopy showing that oxygen exposure converts an active [4Fe-4S] cluster to an inactive [3Fe-4S] cluster. Our anaerobically conducted kinetic studies reveal a preference for fumarate over S-malate. Our data further reveal that single alanine substitutions of T467, R421, R471, D135, and H334 decrease kcat values 9-16000-fold without substantially affecting Km values, suggesting that these residues function in catalytic roles. Crystal structures of LmFH-2 variants are consistent with this idea, showing similar bidentate binding to the unique iron of the [4Fe-4S] cluster for substrate S-malate as observed in wild type FH. We further present LmFH-2 structures with substrate fumarate and weak inhibitors succinate and malonate bound in the active site and the first structure of an LmFH that is substrate-free and inhibitor-free, the latter showing increased mobility in the C-terminal domain. Collectively, these data provide insight into the molecular basis for the reaction catalyzed by LmFHs, enzymes that are potential drug targets against leishmaniasis. Structural and Biochemical Investigations of the [4Fe-4S] Cluster-Containing Fumarate Hydratase from Leishmania major.,Feliciano PR, Drennan CL Biochemistry. 2019 Nov 27. doi: 10.1021/acs.biochem.9b00923. PMID:31743022[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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