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

## TL;DR

Scientists modified an enzyme called HphA to change how it interacts with amino acids, potentially improving the stability and availability of peptides.

## Contribution

The study identifies residue A73 in HphA as key to substrate specificity and shows that mutating it relaxes the enzyme's selectivity.

## Key findings

- The A73L mutation in HphA significantly broadened its substrate specificity.
- HphA A73L showed activity with multiple substrates including l-Tyr, l-Val, l-Leu, l-Ser, l-Trp, and l-Asp.
- Kinetic assays confirmed that the A73L mutation made HphA more flexible in substrate use compared to the wild type.

## Abstract

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.