Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation.
Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation.
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DOI：
10.1016/j.isci.2023.106902
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发表时间：
2023-06-16
期刊：
影响因子：
5.8
通讯作者：
Drennan, Catherine L.
中科院分区：
文献类型：
--
作者：
Andorfer, Mary C.;King-Roberts, Devin T.;Imrich, Christa N.;Brotheridge, Balyn G.;Drennan, Catherine L.
烃类的厌氧微生物降解通常是通过x -琥珀酸合酶（xss）将烃类加到富马酸盐中来启动的。xss使用由激活酶（XSS-AE）安装的甘酰基自由基辅因子来催化这种碳-碳偶联反应。虽然活化步骤对催化至关重要，但由于XSS-AEs的不溶性，以前不可能在体外进行。本研究采用基因组挖掘方法，找到了一种可在大肠杆菌中溶性表达的4-异丙基苄基琥珀酸合酶(IBSS)-AE （IbsAE）。这种可溶的XSS-AE可以在体外激活IBSS和已被广泛研究的苄基琥珀酸合成酶（BSS），使我们能够从生物化学角度探索xss。首先，我们研究了BSS亚基的作用，发现β亚基加速了碳氢化合物的加成速率。展望未来，这里收集的方法和见解可以更广泛地用于理解和设计xss作为合成有用的生物催化剂。可溶性XSS激活酶（XSS- ae）是通过基因组挖掘发现的一种可溶的XSS- ae，它允许体外甘酰基在XSS中安装。生物催化;结构生物学;生物工程
Anaerobic microbial degradation of hydrocarbons is often initiated through addition of the hydrocarbon to fumarate by enzymes known as X-succinate synthases (XSSs). XSSs use a glycyl radical cofactor, which is installed by an activating enzyme (XSS-AE), to catalyze this carbon-carbon coupling reaction. The activation step, although crucial for catalysis, has not previously been possible in vitro because of insolubility of XSS-AEs. Here, we take a genome mining approach to find an XSS-AE, a 4-isopropylbenzylsuccinate synthase (IBSS)-AE (IbsAE) that can be solubly expressed in Escherichia coli. This soluble XSS-AE can activate both IBSS and the well-studied benzylsuccinate synthase (BSS) in vitro, allowing us to explore XSSs biochemically. To start, we examine the role of BSS subunits and find that the beta subunit accelerates the rate of hydrocarbon addition. Looking forward, the methodology and insight gathered here can be used more broadly to understand and engineer XSSs as synthetically useful biocatalysts. X-succinate synthases (XSSs) use a glycyl radical to functionalize hydrocarbons Soluble XSS activating enzymes (XSS-AEs) were found through genome mining A soluble XSS-AE allowed in vitro glycyl radical installation within XSSs An auxiliary subunit is critical for fumarate hydroalkylation using XSSs Biochemistry; Biocatalysis; Structural biology; Bioengineering
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影响因子：
15
作者：
Byer AS;Yang H;McDaniel EC;Kathiresan V;Impano S;Pagnier A;Watts H;Denler C;Vagstad AL;Piel J;Duschene KS;Shepard EM;Shields TP;Scott LG;Lilla EA;Yokoyama K;Broderick WE;Hoffman BM;Broderick JB
通讯作者：
Broderick JB
影响因子：
15
作者：
Qiao, CH;Marsh, ENG
通讯作者：
Marsh, ENG
影响因子：
4.8
作者：
Peng, Yi;Veneziano, Susan E.;Broderick, Joan B.
通讯作者：
Broderick, Joan B.
DOI：
10.1073/pnas.81.5.1332
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
KNAPPE, J;NEUGEBAUER, FA;GANZLER, M
通讯作者：
GANZLER, M
影响因子：
7.7
作者：
Backman, Lindsey R. F.;Huang, Yolanda Y.;Drennan, Catherine L.
通讯作者：
Drennan, Catherine L.