Improved selectivity of an engineered multi-product terpene synthase.
Improved selectivity of an engineered multi-product terpene synthase.
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DOI：
10.1039/c4ob00479e
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发表时间：
2014-05
影响因子：
3.2
通讯作者：
Ryan Lauchli;Julia Pitzer;Rebekah Z. Kitto;K. Z. Kalbarczyk;Kersten S. Rabe
中科院分区：
文献类型：
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作者：
Ryan Lauchli;Julia Pitzer;Rebekah Z. Kitto;K. Z. Kalbarczyk;Kersten S. Rabe
利用非天然底物对倍半萜合成酶Cop2进行了高通量筛选，以测定其环化活性。鉴定出Cop2突变体含有三个氨基酸取代。该突变体17H2以77%的选择性将天然底物FPP转化为锗烯d -4-醇。这种选择性与母酶形成对比，母酶的产物混合物中产生了29%的锗烯d -4-醇和46%的α-二醇。结果表明，这些突变都对这种选择性有贡献，同源性模型表明，这些突变位于活性位点附近，但不太可能成为合理方法的突变目标。动力学比较表明，17H2的kcat/KM为0.62 mM(-1) s(-1)，与亲本Cop2的kcat/KM为0.58 mM(-1) s（-1）基本一致。
Mutation of the sesquiterpene synthase Cop2 was conducted with a high-throughput screen for the cyclization activity using a non-natural substrate. A mutant of Cop2 was identified that contained three amino acid substitutions. This mutant, 17H2, converted the natural substrate FPP into germacrene D-4-ol with 77% selectivity. This selectivity is in contrast to that of the parent enzyme in which germacrene D-4-ol is produced as 29% and α-cadinol is produced as 46% of the product mixture. The mutations were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a kcat/KM of 0.62 mM(-1) s(-1), which is nearly identical to that of the parent Cop2, which had a kcat/KM of 0.58 mM(-1) s(-1).