# Characterizing and Tailoring the Substrate Profile of a γ-Glutamyltransferase Variant

## TL;DR

This paper explores a synthetic transport system to improve the delivery of noncanonical molecules into cells and enhances the enzyme GGTxe to better release cargo molecules.

## Contribution

The study introduces a directed evolution approach to improve GGTxe activity for amino acid conjugates and identifies a key residue for cargo unloading.

## Key findings

- Vector-amino acid conjugates were poorly hydrolyzed by GGTxe.
- Directed evolution improved GGTxe activity for these conjugates.
- Residue D386 was found to be important for cargo unloading.

## Abstract

Xenobiology is an emerging field that focuses on the
extension
and redesign of biological systems through the use of laboratory-derived
xenomolecules, which are molecules that are new to the metabolism
of the cell. Despite the enormous potential of using xenomolecules
in living organisms, most noncanonical building blocks still need
to be supplied externally, and often poor uptake into cells limits
wider applicability. To improve the cytosolic availability of noncanonical
molecules, a synthetic transport system based on portage transport
was developed, in which molecules of interest “cargo”
are linked to a synthetic transport vector that enables piggyback
transport through the alkylsulfonate transporter (SsuABC) of Escherichia coli. Upon cytosolic delivery, the vector-cargo
conjugate is enzymatically cleaved by GGTxe, leading to
the release of the cargo molecule. To deepen our understanding of
the synthetic transport system, we focused on the characterization
and further development of the enzymatic cargo release step. Hence,
the substrate scope of GGTxe was characterized using a
library of structurally diverse vector-cargo conjugates and MS/MS-based
quantification of hydrolysis products in a kinetic manner. The resulting
substrate tolerance characterization revealed that vector-amino acid
conjugates were significantly unfavored. To overcome this shortcoming,
a selection system based on metabolic auxotrophy complementation and
directed evolution of GGTxe was established. In a directed
evolution campaign, we improved the enzymatic activity of GGTxe for vector-amino acid conjugates and revealed the importance
of residue D386 in the cargo unloading step.