A catalytic dyad modulates conformational change in the CO(2)-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase.
A catalytic dyad modulates conformational change in the CO(2)-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase.
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DOI：
10.1016/j.jbc.2022.101884
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发表时间：
2022-05
影响因子：
4.8
通讯作者：
Bothner, Brian
中科院分区：
文献类型：
--
作者：
Mattice, Jenna R.;Shisler, Krista A.;DuBois, Jennifer L.;Peters, John W.;Bothner, Brian
2-酮丙基辅酶M氧化还原酶/羧化酶（2-KPCC）是含有黄素和半胱氨酸二硫的氧化还原酶家族（DSOR）的成员，它催化大气CO2与酮/烯酸酯亲核试剂之间的独特反应生成乙酰乙酸。然而，这种反应的机理尚不清楚。在这里，我们提出的证据表明，2-KPCC，与已被充分表征的DSOR酶谷胱甘肽还原酶相反，在催化过程中会发生构象变化。在底物和抑制剂存在的情况下，我们使用一套生物物理技术，包括有限蛋白水解、差示扫描荧光法和天然质谱法，观察了催化循环中不同配体结合的2-KPCC物种之间的构象差异。位点特异性氨基酸变异分析表明，活性位点内的2- kpcc定义残基Phe501-His506对于这些配体诱导的构象变化的转导很重要。我们认为这些构象变化促进了H+和CO2之间的底物区分，从而有利于代谢首选的羧化产物乙酰乙酸。
2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO2 and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H+ and CO2 to favor the metabolically preferred carboxylation product, acetoacetate.
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影响因子：
4.8
作者：
Streit, Bennett R.;Mattice, Jenna R.;DuBois, Jennifer L.
通讯作者：
DuBois, Jennifer L.
影响因子：
3.7
作者：
Bothner, B;Taylor, D;Johnson, JE
通讯作者：
Johnson, JE
影响因子：
2.9
作者：
Nocek, B;Jang, SB;Peters, JW
通讯作者：
Peters, JW
影响因子：
3.5
作者：
Pandey, Arti S.;Mulder, David W.;Peters, John W.
通讯作者：
Peters, John W.
DOI：
10.1016/j.jbc.2021.100961
发表时间：
2021-08
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Prussia GA;Shisler KA;Zadvornyy OA;Streit BR;DuBois JL;Peters JW
通讯作者：
Peters JW