A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation

Abstract

Naturally occurring enzyme homologs often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homolog. We demonstrate that a small set of mutations of the tryptophan synthase β-subunit (TrpB) from Pyrococcus furiosus, which mimic the activation afforded by binding of the α-subunit, has a similar activating effect in TrpB homologs with as little as 57% sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism, mimicry of α-subunit binding. From this collection of stand-alone enzymes, we identified a new catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans, a biologically important class of compounds. This investigation demonstrates how allosteric activation can be recapitulated throughout a protein family to efficiently explore natural sequence diversity for desirable biocatalytic transformations.

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.…”

Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity

“…10 We found that each of these enzymes also performs a productive reaction with IGP and Thr. In the presence of Ser, however, and even with a 1,000-fold molar excess of Thr over Ser, there are at most trace amounts of β-MeTrp formed (Table S1).…”

Engineered Biosynthesis of β‐Alkyl Tryptophan Analogues

“…We previously engineered the β-subunit of the PLPdependent enzyme tryptophan synthase from the thermophilic archaeon Pyrococcus furiosus (PfTrpB) as a stand-alone ncAA synthase able to generate tryptophan (Trp) analogs from serine (Ser) and the corresponding substituted indole (Figure 2a). [23][24][25] Further engineering of PfTrpB for improved C-C bond formation with indole analogs and threonine (Thr) led to PfTrpB 2B9 (eight mutations from wild-type PfTrpB), which exhibited a >1,000-fold improvement in (2S, 3S)-β-methyltryptophan (β-MeTrp) production relative to wild type (Figure 2b). [26,27] While the reactive amino-acrylate intermediate (E (A-A)) (Figure 3a) readily forms with Thr, mechanistic analysis showed that competing hydrolysis of (E(A-A)) resulted in abortive deamination that consumed the amino acid substrate (Figure 3b), [28,29] limiting the enzyme's yield (typically < 50%) with a single equivalent of Thr.…”

“…[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).…”