Synthesis of β-Branched Tryptophan Analogues Using an Engineered Subunit of Tryptophan Synthase

Abstract

We report that l-threonine may substitute for l-serine in the β-substitution reaction of an engineered subunit of tryptophan synthase from Pyrococcus furiosus, yielding (2S,3S)-β-methyltryptophan (β-MeTrp) in a single step. The trace activity of the wild-type β-subunit on this substrate was enhanced more than 1000-fold by directed evolution. Structural and spectroscopic data indicate that this increase is correlated with stabilization of the electrophilic aminoacrylate intermediate. The engineered biocatalyst also reacts with a variety of indole analogues and thiophenol for diastereoselective C-C, C-N, and C-S bond-forming reactions. This new activity circumvents the 3-enzyme pathway that produces β-MeTrp in nature and offers a simple and expandable route to preparing derivatives of this valuable building block.

Figure 1

(a) Condensation of 1 and 2 using wild-type TrpS from different species. (b) Proposed reaction with 4 using engineered variants of TrpB. (c) Examples of natural products with β-MeTrp as a biosynthetic intermediate.

Figure 2

Proposed catalytic cycle showing the expected UV-vis absorbance peaks for key intermediates. Detailed discussion of the TrpS mechanism is given in ref. .

Figure 3

Productivity of 5 with heat-treated lysates of E. coli expressing PfTrpB enzymes. Productivity is defined as the amount of 5 formed after 90 minutes at 75 °C from a starting reaction mixture containing 20 mM of 1 and 4.

Figure 4

(a) The co-crystal structure of PfTrpB^WT with 4 shows that Thr binds non-covalently. Mesh corresponds to Fo-Fc omit map displayed at 2.5 σ. (b) The UV-vis spectrum of PfTrpB^2B9 (black) shows a peak at 412 nm, corresponding to E(Ain). Addition of 20 mM 4 (red) has a mixed population of E(Aex₁) at 428 nm and E(A-A) at ∼350 nm, indicating covalent substrate binding.

Chart 1. Production of β-MeTrp analogs

^aTotal turnover numbers are given in parentheses. Reactions conducted in triplicate. Circles indicate the site of alkylation. ^bSingle reaction conducted at 25 °C with 12.5 mM DTT. See SI for further detail.