Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation
Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation
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DOI：
10.1021/jacs.9b09864
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发表时间：
2019-12-18
影响因子：
15
通讯作者：
Arnold, Frances H.
中科院分区：
文献类型：
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作者：
Dick, Markus;Sarai, Nicholas S.;Arnold, Frances H.
我们之前设计了色氨酸合成酶（TrpB）的β亚基，它催化l -丝氨酸和吲哚缩合成l -色氨酸，从l -丝氨酸和吲哚衍生物或其他亲核试剂合成一系列非规范氨基酸。在这里，我们采用定向进化来设计TrpB，使其接受3-取代的氧吲哚并形成C-C键，从而形成新的四元立体中心。最初，可以使用3-取代吲哚的变体优先在底物的N-1上形成N-C键。保护N-1促进了向c -烷基化的进化，当保护被移除时，这种进化会持续下去。经过6代定向进化，TrpB - Pf（quat）的3-取代吲哚烷基化活性提高了400倍，并且能够在氨基酸产物的γ位置选择性地形成新的全碳季位立体中心。该酶还能在结构相似的内酯和酮上烷基化并形成全碳的季元立体中心，对叔碳具有优异的区域选择性。通过微晶电子衍射（MicroED）测定了两种产物的γ -立体中心构型，并报道了使用猎鹰III直接电子探测器获得的一个小分子的MicroED结构。TrpB - Pf（quat）具有高耐热性，在> - 500mg /L大肠杆菌培养中表达，为构建具有全碳四元立体中心的多种非规范氨基酸提供了高效、可持续和选择性的平台。
We previously engineered the beta-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-1 of the substrate. Protecting N-1 encouraged evolution toward C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pf(quat) 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 gamma-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 gamma-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 E. coli culture, TrpB Pf(quat) offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.