Lauchli Ryan, Pitzer Julia, Kitto Rebekah Z, Kalbarczyk Karolina Z, Rabe Kersten S
California Institute of Technology, Division of Chemistry & Chemical Engineering, 1200 E. California Blvd., Pasadena CA 91125, USA.
Org Biomol Chem. 2014 Jun 21;12(23):4013-20. doi: 10.1039/c4ob00479e.
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).
利用非天然底物对倍半萜合酶Cop2进行突变，通过高通量筛选其环化活性。鉴定出一个Cop2突变体，该突变体包含三个氨基酸替换。这个名为17H2的突变体将天然底物法尼基焦磷酸（FPP）转化为杜松烯D - 4 -醇的选择性为77%。这种选择性与亲本酶不同，亲本酶产生的杜松烯D - 4 -醇占产物混合物的29%，α - 杜松醇占46%。研究表明，这些突变各自对这种选择性有贡献，并且一个同源模型表明这些突变位于活性位点附近，不过通过合理方法不太可能将其作为突变靶点。动力学比较表明，17H2的催化常数与米氏常数之比（kcat/KM）为0.62 mM⁻¹ s⁻¹，这与亲本Cop2的kcat/KM（0.58 mM⁻¹ s⁻¹）几乎相同。
