The unique Phe-His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S-C bond cleavage.
The unique Phe-His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S-C bond cleavage.
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DOI：
10.1016/j.jbc.2021.100961
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发表时间：
2021-08
期刊：
影响因子：
--
通讯作者：
Peters JW
中科院分区：
文献类型：
--
作者：
Prussia GA;Shisler KA;Zadvornyy OA;Streit BR;DuBois JL;Peters JW
2-酮丙基-辅酶M氧化还原酶/羧化酶（2-KPCC）酶是二硫化物氧化还原酶（DSOR）家族中唯一具有羧化活性的成员，其对于还原性裂解S-S键是重要的。2-KPCC催化2-酮丙基辅酶M转化为乙酰乙酸，乙酰乙酸用作碳源，在受控反应中排除质子。DSOR中存在的保守His-Glu基序是质子化步骤的关键;然而，在2-KPCC中，二联体被Phe-His取代。在这里，我们建议，这种差异是重要的耦合羧化与C-S键裂解。我们将2-KPCC中的Phe-His二联体替换为更像DSOR的二联体，用组氨酸（F501 H）替换苯丙氨酸，用谷氨酸（H506 E）替换组氨酸，并解析了F501 H和双变体F501H_H506E的晶体结构。我们发现F501保护烯醇丙酮中间体免受质子的影响，并且F501 H变体强烈促进质子化。我们还提供了参与H506残基在乙酰乙酸形成过程中稳定显影电荷的证据，乙酰乙酸在WT中而不是H506 E变体酶中充当产物抑制剂。最后，我们确定F501 H取代促进了与黄素腺嘌呤二核苷酸的DSOR样电荷转移相互作用，消除了对半胱氨酸作为内部碱基的需要。总之，这些结果表明，2-KPCC二联体负责选择性地促进羧化和抑制质子化形成乙酰乙酸。
The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S–S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His–Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe–His. Here, we propose that this difference is important for coupling carboxylation with C–S bond cleavage. We substituted the Phe–His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.
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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
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：
3.5
作者：
Pandey, Arti S.;Mulder, David W.;Peters, John W.
通讯作者：
Peters, John W.
影响因子：
64.8
作者：
SCHULZ, GE;SCHIRMER, RH;PAI, EF
通讯作者：
PAI, EF
影响因子：
2.9
作者：
Nocek, B;Jang, SB;Peters, JW
通讯作者：
Peters, JW