Unlocking Reactivity of TrpB: A General Biocatalytic Platform for Synthesis of Tryptophan Analogues

Abstract

Derivatives of the amino acid tryptophan (Trp) serve as precursors for the chemical and biological synthesis of complex molecules with a wide range of biological properties. Trp analogs are also valuable as building blocks for medicinal chemistry and as tools for chemical biology. While the enantioselective synthesis of Trp analogs is often lengthy and requires the use of protecting groups, enzymes have the potential to synthesize such products in fewer steps and with the pristine chemo- and stereoselectivity that is a hallmark of biocatalysis. The enzyme TrpB is especially attractive because it can form Trp analogs directly from serine (Ser) and the corresponding indole analog. However, many potentially useful substrates, including bulky or electron-deficient indoles, are poorly accepted. We have applied directed evolution to TrpB from Pyrococcus furiosus and Thermotoga maritima to generate a suite of catalysts for the synthesis of previously intractable Trp analogs. For the most challenging substrates, such as nitroindoles, the key to improving activity lay in the mutation of a universally conserved and mechanistically important residue, E104. The new catalysts express at high levels (>200 mg/L of E. coli culture) and can be purified by heat treatment; they can operate up to 75 °C (where solubility is enhanced) and can synthesize enantiopure tryptophan analogs substituted at the 4-, 5-, 6-, and 7-positions, using Ser and readily available indole analogs as starting materials. Spectroscopic analysis shows that many of the activating mutations suppress the decomposition of the active electrophilic intermediate, an amino-acrylate, which aids in unlocking the synthetic potential of TrpB.

Facile in Vitro Biocatalytic Production of Diverse Tryptamines

“…Many substituted indoles and even other amino acids can participate in this versatile reaction, which affords access to complex starting materials for downstream modification . Recently, these efforts have been made even easier by evolving the β‐subunit of tryptophan synthase (TrpB; EC 4.2.1.20) for efficient stand‐alone function . These engineered TrpBs can operate at high temperatures, which is helpful in solubilizing hydrophobic indoles, and provide access to highly complex Trp analogues in good yield.…”

“…To test the promiscuity of Rgn TDC, we sought to measure its kinetic parameters with a complete set of 4‐, 5‐, 6‐, and 7‐chlorotryptophans; however, the requisite enantiopure chlorotryptophans were not readily available. We therefore prepared each Trp analogue from its indole precursor through a simple biocatalytic reaction by using engineered TrpBs from Pyrococcus furiosus with high stand‐alone activity . Surprisingly, the kinetic parameters of Rgn TDC with each chlorotryptophan showed only modest decreases in catalytic efficiency when compared to the native Trp substrate (Table , Figure S3).…”

Direct Enzymatic Synthesis of a Deep‐Blue Fluorescent Noncanonical Amino Acid from Azulene and Serine

“…Enzymes can perform enantio‐ and regioselective chemistry in the presence of reactive moieties such as primary amines, obviating the need for expensive and intricate chiral catalysts, chiral separations, and protecting groups. To this end, our lab previously reported the directed evolution of the tryptophan synthase β‐subunit (TrpB) as a stand‐alone biocatalytic platform for the synthesis of diverse tryptophan analogues (Scheme B) . Herein, we report a simple, efficient route for the synthesis of AzAla from stable, commercially available starting materials by using an engineered TrpB (Scheme C).…”

“…The significant effect of the E104(105)G mutation suggested that this conserved catalytic residue might be playing an important role in the non‐native azulene reaction. We explored this possibility by examining two engineered variants with and without the E104(105)G mutation: Pf 5G8, which exhibits optimal activity at 75 °C, and Tm 9D8*, which exhibits optimal activity at lower temperatures such as 37 °C . Challenging the enzymes with indole demonstrated that this mutation only modestly decreases the rate of Trp formation (Figure ), with an additional slight decrease in the chemoselectivity of the reaction that leads to the formation of trace amounts of isoTrp (a product of the N‐alkylation of indole, shown in Figure S2 and described previously).…”

Engineered Biosynthesis of β‐Alkyl Tryptophan Analogues

“…[23][24][25] We performed analytical biotransformations with 11 representative nucleophiles with three β-branched amino acid substrates, yielding 27 tryptophan analogs, 20 of which are previously unreported (Table 2). Each reaction was analyzed by liquid-chromatography/mass spectrometry (LCMS) and TTN were calculated by comparing product and substrate absorption at the isosbestic wavelength (Table S4).…”