Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Abstract

We previously engineered the tryptophan synthase b-subunit (TrpB), which catalyzes the condensation reaction between L-serine and indole to form L-tryptophan, to synthesize a range of modified tryptophans from serine and indole derivatives. In this study, we used directed evolution to engineer TrpB to accept 3-substituted oxindoles and form CC bonds leading to new quaternary stereocenters. At first, the TrpBs that could use 3-substituted oxindoles preferentially formed N-C bonds by attacking the oxindole N1 atom. We found, however, that protecting the nitrogen encouraged evolution towards C-alkylation, which persisted even when this protection was removed. After seven rounds of evolution leading to a 400-fold improvement in activity, variant Pfquat efficiently alkylates 3-substituted oxindoles to selectively form new stereocenters at the γ-position of the amino acid products. The configuration of the new γ-stereocenter of one of the products was determined from the crystal structure obtained by microcrystal electron diffraction (MicroED). Substrates structurally related to 3methyloxindole such as lactones and ketones can also be used by the enzyme for quaternary carbon bond formation, where the biocatalyst exhibits excellent regioselectivity for the tertiary carbon atom. Highly thermostable and expressed at > 500 mg/L E. coli culture, TrpB Pfquat provides an efficient and environmentally-friendly platform for the preparation of noncanonical amino acids bearing quaternary carbons.

Biocatalysis: Enzymatic Synthesis for Industrial Applications

“… Tryptophan synthase (TrpB)‐catalyzed C−C bond formation to produce a) β‐methyl tryptophan derivatives; [193] b) tryptophan derivatives with quaternary carbons [194] …”

Aldehyde catalysis – from simple aldehydes to artificial enzymes

“…This reaction generated new noncanonical amino acids with quaternary carbon stereocenters (Scheme 33). 57 Substrates as lactone and ketone were also compatible with this reaction and formed the quaternary carbon stereocenters. It is also worth noting that by applying optimized engineered TrpB, all products were obtained in enantio-pure fashion and the reaction is highly chemo-selective that no N atom alkylated product was observed for the oxindoles substrates.…”

Biokatalyse: Enzymatische Synthese für industrielle Anwendungen

“…Es wurden Va rianten entwickelt, die Methylserin als Substrat akzeptieren und die Bildung eines b-Methyltryptophan-Derivates (Schema 42 a) erlauben [193] und von 3-substituierten Oxindolen, die zu quartären Zentren am Indol an dieser Bindungsstelle führen (Schema 42 b). [194] 6.1.3. Aldolasen und Nukleosid-Phosphorylasen Enzyme sind bestens fürK askadenreaktionen geeignet, weil sie von Natur aus dafüre ntworfen wurden, unter ähnlichen Bedingungen bezüglich Lçsungsmittel, Te mperaturen und weiteren Parametern zu arbeiten und weil sie schon an sich eine Selektivitätaufweisen, die es erlaubt, dass mehrere Katalysatoren zusammenarbeiten -b ei geringer Gefahr von Kreuzreaktivität.…”