Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 N Torrey Pines Road, La Jolla, California 92037, United States.
Department of Biological Sciences, Bridge Institute, University of Southern California , 3430 S Vermont Avenue, Los Angeles, California 90089, United States.
J Am Chem Soc. 2017 Apr 26;139(16):5728-5731. doi: 10.1021/jacs.7b02273. Epub 2017 Apr 17.
We have engineered the protein interface of the Escherichia coli chorismate mutase (EcCM) homodimer to be dependent on incorporation of a noncanonical amino acid (ncAA) at residue 72. The large hydrophobic amino acid p-benzoyl phenylalanine (pBzF) was substituted for Tyr72, which led to a catalytically inactive protein. A library of five residues (Leu25', Arg29', Leu76, Ile80' and Asp83') surrounding pBzF72 was generated and subjected to a growth based selection in a chorismate mutase deficient strain. An EcCM variant (Phe25', pBzF72, Thr76, Gly80' and Tyr83') forms a stable homodimer, has catalytic activity similar to the wild type enzyme, and unfolds with a T of 53 °C. The X-ray crystal structure reveals a pi-pi stacking and hydrogen bonding interactions that stabilize the new protein interface. The strategy described here should be useful for generating organisms that are dependent on the presence of a ncAA for growth.
我们设计了大肠杆菌分支酸变位酶（EcCM）同源二聚体的蛋白质界面，使其依赖于在 72 位残基掺入非天然氨基酸（ncAA）。将大的疏水性氨基酸对苯甲酰苯丙氨酸（pBzF）取代 Tyr72，导致酶失去催化活性。生成了围绕 pBzF72 的五个残基（Leu25'、Arg29'、Leu76、Ile80'和 Asp83'）的文库，并在分支酸变位酶缺陷型菌株中进行基于生长的选择。EcCM 变体（Phe25'、pBzF72、Thr76、Gly80'和 Tyr83'）形成稳定的同源二聚体，具有与野生型酶相似的催化活性，并且解折叠的 Tm 为 53°C。X 射线晶体结构揭示了一个 pi-pi 堆积和氢键相互作用，稳定了新的蛋白质界面。这里描述的策略应该有助于生成依赖 ncAA 存在才能生长的生物体。
