Non-canonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products and therapeutics. Despite their bioactive potential, applications of β-branched ncAAs are limited by their availability: synthesis requires multi-step routes that often exhibit low overall stereoselectivity. Enzymes use simple materials and offer an efficient and sustainable alternative to chemical synthesis. However, like traditional organic methodologies, the enzymatic synthesis of β-branched ncAAs is often confounded by the presence of a second stereocenter. We previously engineered the β-subunit of the PLP-dependent enzyme tryptophan synthase from Pyrococcus furiosus (PfTrpB) as a stand-alone ncAA synthase able to generate tryptophan analogs from serine or threonine and the corresponding substituted indole. However, the enzyme’s yield and substrate scope were limited by competing hydrolysis of the reactive amino-acrylate intermediate.
The ideal synthase would need to utilize diverse indole and amino acid analogs to produce an array of β-branched ncAAs, making these desirable molecules readily available for the first time. Here we report such an engineered catalyst, PfTrpB7E6, that integrates nine mutations from mechanism-guided engineering, random mutagenesis, and recombination. We demonstrate the utility of PfTrpB7E6 as an ncAA synthase by producing 27 β-branched tryptophan analogs. The molecular basis for the efficient catalysis and versatile substrate scope was explored through X-ray crystallography and UV-visible light spectroscopy, which revealed that a combination of active-site and remote mutations increases the abundance and persistence of a key reactive intermediate. This enzyme provides a simple and environmentally benign platform for preparation of β-branched tryptophans.