Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.protein engineering | allostery | noncanonical amino acid | PLP

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.

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.

An enantioselective total synthesis of the norditerpenoid alkaloid nigelladine A is described. Strategically, the synthesis relies on a late stage C-H oxidation of an advanced intermediate. While traditional chemical methods failed to deliver the desired outcome, an engineered cytochrome P450 enzyme was employed to effect a chemo- and regioselective allylic C–H oxidation in the presence of four oxidizable positions. The enzyme variant was readily identified from a focused library of three enzymes, allowing for completion of the synthesis without the need for extensive screening.

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.