p-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA.
p-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA.
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硝基苯酯为硫醇酶OLEA提供了新的见解和应用。
DOI：
10.1016/j.csbj.2021.05.031
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发表时间：
2021
影响因子：
6
通讯作者：
Wackett LP
中科院分区：
文献类型：
--
作者：
Smith MD;Tassoulas LJ;Biernath TA;Richman JE;Aukema KG;Wackett LP
硫解酶OleA通过酰基辅酶A(CoA)底物启动β-内酯天然产物和膜碳氢化合物的生物合成，但本研究表明对硝基苯基烷酸酯可以替代，对酶机理有了新的认识，并提供了一种更便宜、更可用的起始底物。与硫解酶不同的是，油酸酶将两条长的(≥C8)酰基链结合到结构相反的疏水通道A和B上，进行非脱羧基的克莱森缩合反应，并启动膜碳氢化合物和β-内酯天然产物的生物合成。通过生物信息学和使用对硝基苯基烷酸酯作为替代底物的高通量筛选，Olea现在已经在数百种不同的细菌中被鉴定出来。在本研究中，对-硝基苯酯被用来探索油的反应机理，并首次被证明加入到Claisen缩合产物中。单独的对硝基苯基烷酸酯底物不发生Claisen缩合反应，但对硝基苯酯和CoA硫代酯的共同孵育产生了混合Claisen产物。混合产物反应通过酰基转移从对硝基苯基转移到酶活性部位半胱氨酸，c143。合成了酯化为对硝基苯酚的酰基链，并证明其与酰基辅酶A底物发生Claisen缩合反应，显示出极大地扩展Claisen产物范围的潜力。使用对-硝基苯基1-13C-癸酸酯，通道A结合的硫酯链被证明是Claisen亲核试剂，这是在任何Olea酶中Claisen反应方向性的第一个直接证据。这些结果都提供了对油催化的新见解，并开辟了一条利用廉价和容易合成的对硝基苯酯来制造用于生物技术应用的非天然碳氢化合物和β-内酯天然产品的途径。
The thiolase enzyme OleA initiates the biosynthesis of β-lactone natural products and membrane hydrocarbons via acyl-Coenzyme A (CoA) substrates, but this study showed that p-nitrophenyl alkanoates can substitute, yielding new insights into the enzyme mechanism and providing a cheaper and more available starting substrate. The OleA enzyme is distinct amongst thiolase enzymes in binding two long (≥C8) acyl chains into structurally-opposed hydrophobic channels, denoted A and B, to carry out a non-decarboxylative Claisen condensation reaction and initiate the biosynthesis of membrane hydrocarbons and β-lactone natural products. OleA has now been identified in hundreds of diverse bacteria via bioinformatics and high-throughput screening using p-nitrophenyl alkanoate esters as surrogate substrates. In the present study, p-nitrophenyl esters were used to probe the reaction mechanism of OleA and shown to be incorporated into Claisen condensation products for the first time. p-Nitrophenyl alkanoate substrates alone were shown not to undergo Claisen condensation, but co-incubation of p-nitrophenyl esters and CoA thioesters produced mixed Claisen products. Mixed product reactions were shown to initiate via acyl group transfer from a p-nitrophenyl carrier to the enzyme active site cysteine, C143. Acyl chains esterified to p-nitrophenol were synthesized and shown to undergo Claisen condensation with an acyl-CoA substrate, showing potential to greatly expand the range of possible Claisen products. Using p-nitrophenyl 1-13C-decanoate, the Channel A bound thioester chain was shown to act as the Claisen nucleophile, representing the first direct evidence for the directionality of the Claisen reaction in any OleA enzyme. These results both provide new insights into OleA catalysis and open a path for making unnatural hydrocarbon and β-lactone natural products for biotechnological applications using cheap and easily synthesized p-nitrophenyl esters.
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影响因子：
15
作者：
Arora, P;Vats, A;Gokhale, RS
通讯作者：
Gokhale, RS
DOI：
10.5195/jmla.2018.515
发表时间：
2018-10-01
期刊：
Journal of the Medical Library Association : JMLA
影响因子：
--
作者：
Gabrielson SW
通讯作者：
Gabrielson SW
影响因子：
2.9
作者：
Goblirsch, Brandon R.;Frias, Janice A.;Wilmot, Carrie M.
通讯作者：
Wilmot, Carrie M.
影响因子：
2.7
作者：
KAWAGUCHI, A;YOSHIMURA, T;OKUDA, S
通讯作者：
OKUDA, S
影响因子：
15
作者：
Engstrom, Karin;Nyhlen, Jonas;Backvall, Jan-E.
通讯作者：
Backvall, Jan-E.