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 1,000-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 analogs 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.

Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of the intermediates shifts to favor covalently bound Trp adducts, which have increased thermodynamic stability. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High-resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a subdomain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.

Tryptophan synthase (TrpS) catalyzes the final steps in the biosynthesis of L-tryptophan from L-serine (Ser) and indole-3-glycerol phosphate (IGP). We report that native TrpS can also catalyze a productive reaction with L-threonine (Thr), leading to (2S,3S)-β-methyltryptophan. Surprisingly, β-substitution occurs in vitro with a 3.4-fold higher catalytic efficiency for Ser over Thr using saturating indole, despite >82,000-fold preference for Ser in direct competition using IGP. Structural data identify a novel product binding site and kinetic experiments clarify the atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle.

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