6vz9

From Proteopedia

(Difference between revisions)

Current revision

Structure of proline utilization A with the FAD covalently modified by L-thiazolidine-2-carboxylate

Structural highlights

6vz9 is a 2 chain structure with sequence from Sinorhizobium meliloti SM11. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.52Å
Ligands:	, , , , , ,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

F7X6I3_SINMM Oxidizes proline to glutamate for use as a carbon and nitrogen source.[PIRNR:PIRNR000197]

Publication Abstract from PubMed

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of L-proline to 1-pyrroline-5-carboxylate. PRODH has emerged as a possible cancer therapy target, and thus the inhibition of PRODH is of interest. Here we show that the proline analog thiazolidine-2-carboxylate (T2C) is a mechanism-based inactivator of PRODH. Structures of the bifunctional proline catabol-ic enzyme proline utilization A (PutA) determined from crystals grown in the presence of T2C feature strong electron density for a 5-membered ring species resembling L-T2C covalently bound to the N5 of the FAD in the PRODH domain. The modified FAD exhibits a large butterfly bend angle, indicating that the FAD is locked into the 2-electron reduced state. Reduction of the FAD is consistent with the crystals lacking the distinctive yellow color of the oxidized enzyme and stopped-flow kinetic data showing that T2C is a sub-strate for the PRODH domain of PutA. A mechanism is proposed in which PRODH catalyzes the oxidation of T2C at the C atom adjacent to the S atom of the thiazolidine ring (C5). Then, the N5 atom of the reduced FAD attacks the C5 of the oxidized T2C species, resulting in the covalent adduct observed in the crystal structure. To our knowledge, this is the first report of T2C inactivating (or inhibiting) PRODH or any other flavoenzyme. These results may inform the design of new mechanism-based inactivators of PRODH for use as chemical probes to study the roles of proline metabolism in cancer.

Covalent Modification of the Flavin in Proline Dehydrogenase by Thiazolidine-2-Carboxylate.,Campbell AC, Becker DF, Gates KS, Tanner JJ ACS Chem Biol. 2020 Mar 11. doi: 10.1021/acschembio.9b00935. PMID:32159324^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Campbell AC, Becker DF, Gates KS, Tanner JJ. Covalent Modification of the Flavin in Proline Dehydrogenase by Thiazolidine-2-Carboxylate. ACS Chem Biol. 2020 Mar 11. doi: 10.1021/acschembio.9b00935. PMID:32159324 doi:http://dx.doi.org/10.1021/acschembio.9b00935

1 Structural highlights
2 Function
3 Publication Abstract from PubMed
4 See Also
5 References

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/6vz9"

Categories: Large Structures | Sinorhizobium meliloti SM11 | Campbell AC | Tanner JJ

@@ Line 3: / Line 3: @@
 <StructureSection load='6vz9' size='340' side='right'caption='[[6vz9]], [[Resolution|resolution]] 1.52&Aring;' scene=''>
 == Structural highlights ==
-<table><tr><td colspan='2'>[[6vz9]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Sinmm Sinmm]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6VZ9 OCA]. For a <b>guided tour on the structure components</b> use [http://proteopedia.org/fgij/fg.htm?mol=6VZ9 FirstGlance]. <br>
+<table><tr><td colspan='2'>[[6vz9]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Sinorhizobium_meliloti_SM11 Sinorhizobium meliloti SM11]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6VZ9 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6VZ9 FirstGlance]. <br>
-</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=FAD:FLAVIN-ADENINE+DINUCLEOTIDE'>FAD</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=NAD:NICOTINAMIDE-ADENINE-DINUCLEOTIDE'>NAD</scene>, <scene name='pdbligand=PEG:DI(HYDROXYETHYL)ETHER'>PEG</scene>, <scene name='pdbligand=PGE:TRIETHYLENE+GLYCOL'>PGE</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene>, <scene name='pdbligand=T2C:(2S)-1,3-thiazolidine-2-carboxylic+acid'>T2C</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]] 1.52&#8491;</td></tr>
-<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[6ufp|6ufp]]</td></tr>
+<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=FAD:FLAVIN-ADENINE+DINUCLEOTIDE'>FAD</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=NAD:NICOTINAMIDE-ADENINE-DINUCLEOTIDE'>NAD</scene>, <scene name='pdbligand=PEG:DI(HYDROXYETHYL)ETHER'>PEG</scene>, <scene name='pdbligand=PGE:TRIETHYLENE+GLYCOL'>PGE</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene>, <scene name='pdbligand=T2C:(2S)-1,3-thiazolidine-2-carboxylic+acid'>T2C</scene></td></tr>
-<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">putA, SM11_chr0102 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=707241 SINMM])</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=6vz9 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6vz9 OCA], [https://pdbe.org/6vz9 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6vz9 RCSB], [https://www.ebi.ac.uk/pdbsum/6vz9 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6vz9 ProSAT]</span></td></tr>
-<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://proteopedia.org/fgij/fg.htm?mol=6vz9 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6vz9 OCA], [http://pdbe.org/6vz9 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6vz9 RCSB], [http://www.ebi.ac.uk/pdbsum/6vz9 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6vz9 ProSAT]</span></td></tr>
 </table>
 == Function ==
-[[http://www.uniprot.org/uniprot/F7X6I3_SINMM F7X6I3_SINMM]] Oxidizes proline to glutamate for use as a carbon and nitrogen source.[PIRNR:PIRNR000197]
+[https://www.uniprot.org/uniprot/F7X6I3_SINMM F7X6I3_SINMM] Oxidizes proline to glutamate for use as a carbon and nitrogen source.[PIRNR:PIRNR000197]
 <div style="background-color:#fffaf0;">
 == Publication Abstract from PubMed ==
-Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, beta-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Delta(1)-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD(+) and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231-491 M(-1) s(-1). The reaction is reversible, and nuclear magnetic resonance characterization ((1)H, (13)C, and (31)P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD(+). We also report a 1.45 A resolution crystal structure of short-chain dehydrogenase/reductase with the NADP(+)-TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P)(+). Because NAD(P)(+)-dependent oxidoreductases are widespread in nature, our results may be broadly relevant.
+Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of L-proline to 1-pyrroline-5-carboxylate. PRODH has emerged as a possible cancer therapy target, and thus the inhibition of PRODH is of interest. Here we show that the proline analog thiazolidine-2-carboxylate (T2C) is a mechanism-based inactivator of PRODH. Structures of the bifunctional proline catabol-ic enzyme proline utilization A (PutA) determined from crystals grown in the presence of T2C feature strong electron density for a 5-membered ring species resembling L-T2C covalently bound to the N5 of the FAD in the PRODH domain. The modified FAD exhibits a large butterfly bend angle, indicating that the FAD is locked into the 2-electron reduced state. Reduction of the FAD is consistent with the crystals lacking the distinctive yellow color of the oxidized enzyme and stopped-flow kinetic data showing that T2C is a sub-strate for the PRODH domain of PutA. A mechanism is proposed in which PRODH catalyzes the oxidation of T2C at the C atom adjacent to the S atom of the thiazolidine ring (C5). Then, the N5 atom of the reduced FAD attacks the C5 of the oxidized T2C species, resulting in the covalent adduct observed in the crystal structure. To our knowledge, this is the first report of T2C inactivating (or inhibiting) PRODH or any other flavoenzyme. These results may inform the design of new mechanism-based inactivators of PRODH for use as chemical probes to study the roles of proline metabolism in cancer.
-Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD(+)-Dependent Enzymes.,Patel SM, Smith TG, Morton M, Stiers KM, Seravalli J, Mayclin SJ, Edwards TE, Tanner JJ, Becker DF Biochemistry. 2020 Aug 28. doi: 10.1021/acs.biochem.0c00490. PMID:32841567<ref>PMID:32841567</ref>
+Covalent Modification of the Flavin in Proline Dehydrogenase by Thiazolidine-2-Carboxylate.,Campbell AC, Becker DF, Gates KS, Tanner JJ ACS Chem Biol. 2020 Mar 11. doi: 10.1021/acschembio.9b00935. PMID:32159324<ref>PMID:32159324</ref>
 From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
 </div>
 <div class="pdbe-citations 6vz9" style="background-color:#fffaf0;"></div>
+==See Also==
+*[[Proline utilization A|Proline utilization A]]
 == References ==
 <references/>
@@ Line 25: / Line 27: @@
 </StructureSection>
 [[Category: Large Structures]]
-[[Category: Sinmm]]
+[[Category: Sinorhizobium meliloti SM11]]
-[[Category: Campbell, A C]]
+[[Category: Campbell AC]]
-[[Category: Tanner, J J]]
+[[Category: Tanner JJ]]
-[[Category: Bifunctional enzyme]]
-[[Category: Flavoenzyme]]
-[[Category: Oxidoreductase]]
-[[Category: Proline catabolism]]
-[[Category: Rossmann fold]]
-[[Category: Substrate channeling]]

6vz9

From Proteopedia

Current revision

Structure of proline utilization A with the FAD covalently modified by L-thiazolidine-2-carboxylate

Structural highlights

Function

Publication Abstract from PubMed

See Also

References

Contents

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