posted on 2024-01-06, 14:06authored byShelby
L. Hooe, Meghna Thakur, Guillermo Lasarte-Aragonés, Joyce C. Breger, Scott A. Walper, Igor L. Medintz, Gregory A. Ellis
Evolution has gifted enzymes with the ability to synthesize
an
abundance of small molecules with incredible control over efficiency
and selectivity. Central to an enzyme’s role is the ability
to selectively catalyze reactions in the milieu of chemicals within
a cell. However, for chemists it is often desirable to extend the
substrate scope of reactions to produce analogue(s) of a desired product
and therefore some degree of enzyme promiscuity is often desired.
Herein, we examine this dichotomy in the context of the violacein
biosynthetic pathway. Importantly, we chose to interrogate this pathway
with tryptophan analogues in vitro, to mitigate possible interference
from cellular components and endogenous tryptophan. A total of nine
tryptophan analogues were screened for by analyzing the substrate
promiscuity of the initial enzyme, VioA, and compared to the substrate
tryptophan. These results suggested that for VioA, substitutions at
either the 2- or 4-position of tryptophan were not viable. The seven
analogues that showed successful substrate conversion by VioA were
then applied to the five enzyme cascade (VioABEDC) for the production
of violacein, where l-tryptophan and 6-fluoro-l-tryptophan
were the only substrates which were successfully converted to the
corresponding violacein derivative(s). However, many of the other
tryptophan analogues did convert to various substituted intermediaries.
Overall, our results show substrate promiscuity with the initial enzyme,
VioA, but much less for the full pathway. This work demonstrates the
complexity involved when attempting to analyze substrate analogues
within multienzymatic cascades, where each enzyme involved within
the cascade possesses its own inherent promiscuity, which must be
compatible with the remaining enzymes in the cascade for successful
formation of a desired product.