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 analogues are also valuable as building blocks for medicinal chemistry and as tools for chemical biology. While the enantioselective synthesis of Trp analogues 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 analogues directly from serine (Ser) and the corresponding indole analogue. 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 analogues. 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 Escherichia coli culture) and can be purified by heat treatment; they can operate up to 75 °C (where solubility is enhanced) and can synthesize enantiopure Trp analogues substituted at the 4-, 5-, 6-, and 7-positions, using Ser and readily available indole analogues 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.