Fig. 4: SUMS-based engineering of the β-subunit of Pyrococcus furiosus tryptophan synthase (PfTrpB).
From: Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis
a General reaction scheme for PfTrpB. Indole analogs were used to screen PfTrpB libraries. The nucleophilic atom is shown with a circle. R = halo, alkyl, nitro, ether, etc. See ref. 47 for a detailed scope of engineered TrpB enzymes. b SUMS results for generally activated variants detected during globally random mutagenesis library screening. Complete library results and experimental conditions are shown in Supplementary Figs. 23–25. c Comparison of Trp and DIT production under competition and single substrate reaction conditions, using purified 2B9 and H275R enzymes. Indoline was present at 10-fold excess (15 mM indoline, 1.5 mM indole) for both competition and single substrate reactions. Complete duplicate data and conditions are shown in Supplementary Fig. 28. d H275 is a second-sphere residue that forms hydrogen bonds with neighboring residues, N166, and Y301 (PDB ID: 6AM8)50. e SUMS results for 2B9 and two variants from the H275X SSM library. 4-CN-indole was also included in reactions, but no product was observed. Complete library results are shown in Supplementary Fig. 27. f Product formation in single substrate reactions. Activity of H275R and H275E is shown relative to activity of 2B9 (black dashed line). g Michaelis–Menten parameters for 2B9, H275R, and H275E with either indole or indoline as the nucleophilic substrate. Kinetic data were measured in triplicate, and complete data including error analysis are shown in Supplementary Fig. 29.
