The enzyme pseudooxynicotine amine oxidase from Pseudomonas putida S16 is not an oxidase, but a dehydrogenase.
The enzyme pseudooxynicotine amine oxidase from Pseudomonas putida S16 is not an oxidase, but a dehydrogenase.
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DOI：
10.1016/j.jbc.2022.102251
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发表时间：
2022-08
影响因子：
4.8
通讯作者：
Stull, Frederick
中科院分区：
文献类型：
--
作者：
Choudhary, Vishakha;Wu, Kevin;Zhang, Zhiyao;Dulchavsky, Mark;Barkman, Todd;Bardwell, James C. A.;Stull, Frederick
土壤细菌恶臭假单胞菌S16可以以尼古丁作为唯一的碳源和氮源存活。烟碱氧化还原酶（NicA 2）和假羟烟碱胺氧化酶（Pnao）均为含黄素胺氧化酶家族的成员，催化烟碱催化途径中的前两个步骤。我们的实验室先前已经表明，与其酶家族的其他成员相反，NicA 2实际上是一种脱氢酶，使用细胞色素c蛋白（CycN）作为其电子受体。Pnao的天然电子受体是未知的;然而，在恶臭假单胞菌S16基因组中，Pnao与cycN和nicA 2形成操纵子，这使我们假设Pnao也可能是使用CycN作为其电子受体的脱氢酶。在这里，我们的动力学特性的Pnao的特点，并表明，Pnao是由O2氧化不良，但可以迅速氧化CycN，表明Pnao确实作为一种脱氢酶，使用CycN作为其氧化剂。比较稳态动力学与瞬态动力学实验表明，产品释放主要限制营业额PNAO。我们还解析了Pnao在2.60 nm处的晶体结构，这表明Pnao具有与NicA 2相似的结构折叠。此外，刚性体对接的结构的CycN与Pnao和NicA 2确定了一个潜在的保守的结合位点CycN对这两种酶。两者合计，我们的结果表明，虽然Pnao和NicA 2显示出高度的相似性，含有黄素的胺氧化酶直接使用双氧，这两种酶实际上是脱氢酶。
The soil-dwelling bacterium Pseudomonas putida S16 can survive on nicotine as its sole carbon and nitrogen source. The enzymes nicotine oxidoreductase (NicA2) and pseudooxynicotine amine oxidase (Pnao), both members of the flavin-containing amine oxidase family, catalyze the first two steps in the nicotine catabolism pathway. Our laboratory has previously shown that, contrary to other members of its enzyme family, NicA2 is actually a dehydrogenase that uses a cytochrome c protein (CycN) as its electron acceptor. The natural electron acceptor for Pnao is unknown; however, within the P. putida S16 genome, pnao forms an operon with cycN and nicA2, leading us to hypothesize that Pnao may also be a dehydrogenase that uses CycN as its electron acceptor. Here we characterized the kinetic properties of Pnao and show that Pnao is poorly oxidized by O2, but can be rapidly oxidized by CycN, indicating that Pnao indeed acts as a dehydrogenase that uses CycN as its oxidant. Comparing steady-state kinetics with transient kinetic experiments revealed that product release primarily limits turnover by Pnao. We also resolved the crystal structure of Pnao at 2.60 Å, which shows that Pnao has a similar structural fold as NicA2. Furthermore, rigid-body docking of the structure of CycN with Pnao and NicA2 identified a potential conserved binding site for CycN on these two enzymes. Taken together, our results demonstrate that although Pnao and NicA2 show a high degree of similarity to flavin containing amine oxidases that use dioxygen directly, both enzymes are actually dehydrogenases.
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影响因子：
62.1
作者：
Dempsey, Jillian L.;Winkler, Jay R.;Gray, Harry B.
通讯作者：
Gray, Harry B.
影响因子：
3.9
作者：
Fitzpatrick PF
通讯作者：
Fitzpatrick PF
影响因子：
2.9
作者：
Tararina MA;Xue S;Smith LC;Muellers SN;Miranda PO;Janda KD;Allen KN
通讯作者：
Allen KN
影响因子：
3
作者：
Luft, JR;Collins, RJ;DeTitta, GT
通讯作者：
DeTitta, GT
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K