Division of Chemistry and Chemical Engineering 210-41 , California Institute of Technology , 1200 East California Boulevard , Pasadena , California 91125 , United States.
Howard Hughes Medical Institute, David Geffen School of Medicine, Departments of Biological Chemistry and Physiology , University of California , Los Angeles , California 90095 , United States.
J Am Chem Soc. 2019 Dec 18;141(50):19817-19822. doi: 10.1021/jacs.9b09864. Epub 2019 Dec 6.
We previously engineered the β-subunit of tryptophan synthase (TrpB), which catalyzes the condensation of l-serine and indole to l-tryptophan, to synthesize a range of noncanonical amino acids from l-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds on N of the substrate. Protecting N encouraged evolution toward C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereocenter at the γ-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the γ-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L culture, TrpB offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.
我们之前对色氨酸合酶（TrpB）的β亚基进行了工程改造，该酶催化 l-丝氨酸和吲哚缩合生成 l-色氨酸，可从 l-丝氨酸和吲哚衍生物或其他亲核试剂合成一系列非典型氨基酸。在此，我们采用定向进化技术改造 TrpB，使其能够接受 3-取代的吲哚酮并形成 C-C 键，从而构建新的季碳立体中心。最初，能够利用 3-取代的吲哚酮的变体优先在底物的 N 上形成 N-C 键。保护 N 有利于 C-烷基化的进化，当保护基团被去除时，这种进化仍然存在。经过六轮定向进化，得到了 TrpB，其对 3-取代的吲哚酮的烷基化活性提高了 400 倍，并且能够选择性地在氨基酸产物的 γ-位形成新的全碳季碳立体中心。该酶还可以烷基化并形成结构相似的内酯和酮的全碳季碳立体中心，在这些反应中，它对叔碳具有极好的区域选择性。通过微晶体电子衍射（MicroED）确定了两种产物的 γ-立体中心的构型，我们还报告了使用 Falcon III 直接电子探测器获得的小分子的 MicroED 结构。TrpB 高度热稳定，在培养物中表达量>500mg/L，为构建具有全碳季碳立体中心的各种非典型氨基酸提供了高效、可持续和选择性的平台。
