Amenability of the Gatekeeper Enzyme HphA to Engineering in the Homologation Pathway of l‑Phenylalanine and l‑Tyrosine through Homology-Based Site-Directed Mutagenesis.

Homologation of amino acids, the addition or deletion of a methylene group onto their side chains, has the potential to increase the biostability and bioavailability of peptide natural products. The first enzyme in the homologation pathway, HphA, was previously characterized and is substrate selective. Bioinformatics studies were used to identify amino acids in the active site of HphA that may play a role in substrate selection, by comparison to homologous enzymes, homocitrate synthase (HCS) and 2-isopropylmalate synthase (IPMS). Single-point mutants for five amino acid residues in HphA's active site were created to mimic those found in HCS and IPMS. Their activities were measured via time-course assays with the natural substrates for HCS and IPMS. Residue A73 was identified as important for the substrate specificity of HphA; therefore, six additional mutations were generated and tested with nine substrates with various side chains. The HphA A73L mutant exhibited the highest activity compared to the other mutants, showing activity with counterparts of l-Tyr (HphA natural substrate), l-Val (IPMS natural substrate), l-Leu, l-Ser, l-Trp, and l-Asp. Kinetic assays were performed with HphA A73L using the active substrates and compared with kinetic data from HphA WT, HCS, and IPMS. These results demonstrated that the A73L mutation significantly relaxed the substrate specificity of HphA, indicating its amenability to engineering. This research will serve as the foundation for future metabolic engineering studies of the enzymatic homologation pathway of amino acids.