7khn: Difference between revisions
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The | ==NicA2 variant N462Y/W427Y in complex with (S)-nicotine== | ||
<StructureSection load='7khn' size='340' side='right'caption='[[7khn]], [[Resolution|resolution]] 2.31Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[7khn]] is a 1 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=7KHN OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7KHN 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]] 2.31Å</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=7khn FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7khn OCA], [https://pdbe.org/7khn PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7khn RCSB], [https://www.ebi.ac.uk/pdbsum/7khn PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7khn ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/F8G0P2_PSEP6 F8G0P2_PSEP6] | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
In Pseudomonas putida, the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of (S)-nicotine to N-methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O2 as the oxidizing substrate show that NicA2 has a low apparent Km of 114 nM for (S)-nicotine with a very low apparent turnover number (kcat of 0.006 s(-1)). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7-12-fold), their reductive half-reactions using (S)-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to kcat. X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 A resolution, respectively) revealed no significant active-site rearrangements. A second substrate-binding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O2 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown. | |||
Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2.,Tararina MA, Dam KK, Dhingra M, Janda KD, Palfey BA, Allen KN Biochemistry. 2021 Feb 2;60(4):259-273. doi: 10.1021/acs.biochem.0c00855. Epub, 2021 Jan 19. PMID:33464876<ref>PMID:33464876</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
<div class="pdbe-citations 7khn" 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 18:25, 18 October 2023
NicA2 variant N462Y/W427Y in complex with (S)-nicotineNicA2 variant N462Y/W427Y in complex with (S)-nicotine
Structural highlights
FunctionPublication Abstract from PubMedIn Pseudomonas putida, the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of (S)-nicotine to N-methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O2 as the oxidizing substrate show that NicA2 has a low apparent Km of 114 nM for (S)-nicotine with a very low apparent turnover number (kcat of 0.006 s(-1)). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7-12-fold), their reductive half-reactions using (S)-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to kcat. X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 A resolution, respectively) revealed no significant active-site rearrangements. A second substrate-binding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O2 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown. Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2.,Tararina MA, Dam KK, Dhingra M, Janda KD, Palfey BA, Allen KN Biochemistry. 2021 Feb 2;60(4):259-273. doi: 10.1021/acs.biochem.0c00855. Epub, 2021 Jan 19. PMID:33464876[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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