6c71: Difference between revisions
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The | ==Nicotine Oxidoreductase in Complex with S-nicotine== | ||
<StructureSection load='6c71' size='340' side='right'caption='[[6c71]], [[Resolution|resolution]] 2.65Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[6c71]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_putida_S16 Pseudomonas putida S16]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6C71 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6C71 FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.649Å</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=NCT:(S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE'>NCT</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=6c71 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6c71 OCA], [https://pdbe.org/6c71 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6c71 RCSB], [https://www.ebi.ac.uk/pdbsum/6c71 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6c71 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/F8G0P2_PSEP6 F8G0P2_PSEP6] | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Nicotine oxidoreductase (NicA2) is a bacterial flavoenzyme, which catalyzes the first step of nicotine catabolism by oxidizing S-nicotine into N-methyl-myosmine. It has been proposed as a biotherapeutic for nicotine addiction because of its nanomolar substrate binding affinity. The first crystal structure of NicA2 has been reported, establishing NicA2 as a member of the monoamine oxidase (MAO) family. However, substrate specificity and structural determinants of substrate binding and/or catalysis have not been explored. Herein, analysis of the pH-rate profile, single-turnover kinetics, and binding data establish that pH does not significantly affect the catalytic rate and product release is not rate-limiting. The X-ray crystal structure of NicA2 with S-nicotine refined to 2.65 A resolution reveals a hydrophobic binding site with a solvent exclusive cavity. Hydrophobic interactions predominantly orient the substrate, promoting the binding of a deprotonated species and supporting a hydride-transfer mechanism. Notably, NicA2 showed no activity against neurotransmitters oxidized by the two isoforms of human MAO. To further probe the substrate range of NicA2, enzyme activity was evaluated using a series of substrate analogues, indicating that S-nicotine is the optimal substrate and substitutions within the pyridyl ring abolish NicA2 activity. Moreover, mutagenesis and kinetic analysis of active-site residues reveal that removal of a hydrogen bond between the pyridyl ring of S-nicotine and the hydroxyl group of T381 has a 10-fold effect on KM, supporting the role of this bond in positioning the catalytically competent form of the substrate. Together, crystallography combined with kinetic analysis provides a deeper understanding of this enzyme's remarkable specificity. | |||
Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.,Tararina MA, Xue S, Smith LC, Muellers SN, Miranda PO, Janda KD, Allen KN Biochemistry. 2018 Jul 3;57(26):3741-3751. doi: 10.1021/acs.biochem.8b00384. Epub, 2018 Jun 13. PMID:29812904<ref>PMID:29812904</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
<div class="pdbe-citations 6c71" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Large Structures]] | |||
[[Category: Pseudomonas putida S16]] | |||
[[Category: Allen KN]] | |||
[[Category: Tararina MA]] | |||
Latest revision as of 17:57, 4 October 2023
Nicotine Oxidoreductase in Complex with S-nicotineNicotine Oxidoreductase in Complex with S-nicotine
Structural highlights
FunctionPublication Abstract from PubMedNicotine oxidoreductase (NicA2) is a bacterial flavoenzyme, which catalyzes the first step of nicotine catabolism by oxidizing S-nicotine into N-methyl-myosmine. It has been proposed as a biotherapeutic for nicotine addiction because of its nanomolar substrate binding affinity. The first crystal structure of NicA2 has been reported, establishing NicA2 as a member of the monoamine oxidase (MAO) family. However, substrate specificity and structural determinants of substrate binding and/or catalysis have not been explored. Herein, analysis of the pH-rate profile, single-turnover kinetics, and binding data establish that pH does not significantly affect the catalytic rate and product release is not rate-limiting. The X-ray crystal structure of NicA2 with S-nicotine refined to 2.65 A resolution reveals a hydrophobic binding site with a solvent exclusive cavity. Hydrophobic interactions predominantly orient the substrate, promoting the binding of a deprotonated species and supporting a hydride-transfer mechanism. Notably, NicA2 showed no activity against neurotransmitters oxidized by the two isoforms of human MAO. To further probe the substrate range of NicA2, enzyme activity was evaluated using a series of substrate analogues, indicating that S-nicotine is the optimal substrate and substitutions within the pyridyl ring abolish NicA2 activity. Moreover, mutagenesis and kinetic analysis of active-site residues reveal that removal of a hydrogen bond between the pyridyl ring of S-nicotine and the hydroxyl group of T381 has a 10-fold effect on KM, supporting the role of this bond in positioning the catalytically competent form of the substrate. Together, crystallography combined with kinetic analysis provides a deeper understanding of this enzyme's remarkable specificity. Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.,Tararina MA, Xue S, Smith LC, Muellers SN, Miranda PO, Janda KD, Allen KN Biochemistry. 2018 Jul 3;57(26):3741-3751. doi: 10.1021/acs.biochem.8b00384. Epub, 2018 Jun 13. PMID:29812904[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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