β-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade
β-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade
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DOI：
10.1039/c6ob02539k
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发表时间：
2017-01-07
影响因子：
3.2
通讯作者：
Hoshino, Tsutomu
中科院分区：
文献类型：
--
作者：
Ito, Ryousuke;Nakada, Chika;Hoshino, Tsutomu
β-香树脂素合成酶活性位点残基的许多功能及其催化机制尚不清楚。在此，我们研究了高度保守的Phe 413，Tyr 259，和Trp 257残基的β-香树脂素合成酶的大戟tirucalli的功能。位点特异性突变体F416 V和F416 M显示出与野生型几乎相同的酶活性，表明催化反应不需要π电子。然而，F416 A突变体产生了大量的四环达玛烷骨架，β-香树脂素的产生减少。这表明Phe 416残基位于D-环形成位点附近，并用于将氧化角鲨烯底物正确地定位在反应腔内。另一方面，Tyr 259和Trp 257残基的主要催化相关功能是将它们的p-电子提供给阳离子中间体。Y259 F变体显示出与野生型几乎等同的活性，但脂肪族突变体如Ala、瓦尔和Leu变体显示出显著降低的活性，并产生四环达玛烷骨架，强烈证明Tyr 259残基通过阳离子-π相互作用稳定了baccharenyl仲阳离子。Trp 257的脂肪族变体表现出显著降低的酶活性，并且羽扇豆醇以高生产率生产，表明Trp 257通过阳离子-π相互作用稳定了油酰基阳离子。芳香族苯丙氨酸和酪氨酸突变体表现出高活性，由于其更多的增加π-电子密度相对于脂肪族突变体，但羽扇豆醇产生的显着高产率，除了β-香树脂醇。Trp残基可能是通过CH-pi相互作用与Me-30稳健结合的原因。与Trp相比，Phe和Tyr突变体的π电子密度降低将导致羽扇豆醇的高产量。
Many of the functions of the active site residues in beta-amyrin synthase and its catalytic mechanism remain unclear. Herein, we examined the functions of the highly conserved Phe413, Tyr259, and Trp257 residues in the beta-amyrin synthase of Euphorbia tirucalli. The site-specific mutants F416V and F416M showed nearly the same enzymatic activities as the wild type, indicating that pi-electrons are not needed for the catalytic reaction. However, the F416A mutant yielded a large amount of the tetracyclic dammarane skeleton, with decreased production of beta-amyrin. This indicates that the Phe416 residue is located near the D-ring formation site and works to position the oxidosqualene substrate correctly within the reaction cavity. On the other hand, the major catalysis-related function of the Tyr259 and Trp257 residues is to yield their p-electrons to the cationic intermediates. The Y259F variant showed nearly equivalent activity to that of the wild type, but aliphatic mutants such as the Ala, Val, and Leu variants showed significantly decreased the activity and yielded the tetracyclic dammarane scaffold, strongly demonstrating that the Tyr259 residue stabilizes the baccharenyl secondary cation via cation-pi interaction. The aliphatic variants of Trp257 exhibited remarkably decreased enzymatic activity, and lupeol was produced in a high production ratio, indicating that Trp257 stabilizes the oleanyl cation via cation-pi interaction. The aromatic Phe and Tyr mutants exhibited high activities owing to their more increased pi-electron density relative to that of the aliphatic mutants, but lupeol was produced in a significantly high yield besides beta-amyrin. The Trp residue is likely to be responsible for the robust binding of Me-30 through CH-pi interaction. The decreased pi-electron density of the Phe and Tyr mutants compared to that of Trp would have resulted in the high production of lupeol.