8fsi
From Proteopedia
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== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>[[8fsi]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli_K-12 Escherichia coli K-12]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=8FSI OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=8FSI FirstGlance]. <br> | <table><tr><td colspan='2'>[[8fsi]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli_K-12 Escherichia coli K-12]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=8FSI OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=8FSI FirstGlance]. <br> | ||
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=K:POTASSIUM+ION'>K</scene>, <scene name='pdbligand=SAM:S-ADENOSYLMETHIONINE'>SAM</scene>, <scene name='pdbligand=SF4:IRON/SULFUR+CLUSTER'>SF4</scene></td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.46Å</td></tr> |
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=K:POTASSIUM+ION'>K</scene>, <scene name='pdbligand=SAM:S-ADENOSYLMETHIONINE'>SAM</scene>, <scene name='pdbligand=SF4:IRON/SULFUR+CLUSTER'>SF4</scene></td></tr> | ||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=8fsi FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=8fsi OCA], [https://pdbe.org/8fsi PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=8fsi RCSB], [https://www.ebi.ac.uk/pdbsum/8fsi PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=8fsi ProSAT]</span></td></tr> | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=8fsi FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=8fsi OCA], [https://pdbe.org/8fsi PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=8fsi RCSB], [https://www.ebi.ac.uk/pdbsum/8fsi PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=8fsi ProSAT]</span></td></tr> | ||
</table> | </table> | ||
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<div style="background-color:#fffaf0;"> | <div style="background-color:#fffaf0;"> | ||
== Publication Abstract from PubMed == | == Publication Abstract from PubMed == | ||
| - | Radical S-adenosyl-l-methionine (SAM) enzymes are ubiquitous in nature and carry out a broad variety of difficult chemical transformations initiated by hydrogen atom abstraction. Although numerous radical SAM (RS) enzymes have been structurally characterized, many prove recalcitrant to crystallization needed for atomic-level structure determination using X-ray crystallography, and even those that have been crystallized for an initial study can be difficult to recrystallize for further structural work. We present here a method for computationally engineering previously observed crystallographic contacts and employ it to obtain more reproducible crystallization of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE). We show that the computationally engineered variant binds a typical RS [4Fe-4S](2+/+) cluster that binds SAM, with electron paramagnetic resonance | + | Radical S-adenosyl-l-methionine (SAM) enzymes are ubiquitous in nature and carry out a broad variety of difficult chemical transformations initiated by hydrogen atom abstraction. Although numerous radical SAM (RS) enzymes have been structurally characterized, many prove recalcitrant to crystallization needed for atomic-level structure determination using X-ray crystallography, and even those that have been crystallized for an initial study can be difficult to recrystallize for further structural work. We present here a method for computationally engineering previously observed crystallographic contacts and employ it to obtain more reproducible crystallization of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE). We show that the computationally engineered variant binds a typical RS [4Fe-4S](2+/+) cluster that binds SAM, with electron paramagnetic resonance properties indistinguishable from the native PFL-AE. The variant also retains the typical PFL-AE catalytic activity, as evidenced by the characteristic glycyl radical electron paramagnetic resonance signal observed upon incubation of the PFL-AE variant with reducing agent, SAM, and PFL. The PFL-AE variant was also crystallized in the [4Fe-4S](2+) state with SAM bound, providing a new high-resolution structure of the SAM complex in the absence of substrate. Finally, by incubating such a crystal in a solution of sodium dithionite, the reductive cleavage of SAM is triggered, providing us with a structure in which the SAM cleavage products 5'-deoxyadenosine and methionine are bound in the active site. We propose that the methods described herein may be useful in the structural characterization of other difficult-to-resolve proteins. |
| - | Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage.,Moody JD, Hill S, Lundahl MN, Saxton AJ, Galambas A, Broderick WE, Lawrence CM, Broderick JB J Biol Chem. 2023 | + | Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage.,Moody JD, Hill S, Lundahl MN, Saxton AJ, Galambas A, Broderick WE, Lawrence CM, Broderick JB J Biol Chem. 2023 Jun;299(6):104791. doi: 10.1016/j.jbc.2023.104791. Epub 2023 , May 6. PMID:37156396<ref>PMID:37156396</ref> |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | ||
Current revision
The structure of a crystallizable variant of E. coli pyruvate formate-lyase activating enzyme bound to SAM
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