2 research outputs found
Probing the Donor and Acceptor Substrate Specificity of the γ-Glutamyl Transpeptidase
γ-Glutamyl transpeptidase (GGT) is a two-substrate
enzyme that plays a central role in glutathione metabolism and is
a potential target for drug design. GGT catalyzes the cleavage of
γ-glutamyl donor substrates and the transfer of the γ-glutamyl
moiety to an amine of an acceptor substrate or water. Although structures
of bacterial GGT have revealed details of the protein–ligand
interactions at the donor site, the acceptor substrate site is relatively
undefined. The recent identification of a species-specific acceptor
site inhibitor, OU749, suggests that these inhibitors may be less
toxic than glutamine analogues. Here we investigated the donor and
acceptor substrate preferences of <i>Bacillus anthracis</i> GGT (CapD) and applied computational approaches in combination with
kinetics to probe the structural basis of the enzyme’s substrate
and inhibitor binding specificities and compare them with human GGT.
Site-directed mutagenesis studies showed that the R432A and R520S
variants exhibited 6- and 95-fold decreases in hydrolase activity,
respectively, and that their activity was not stimulated by the addition
of the l-Cys acceptor substrate, suggesting an additional
role in acceptor binding and/or catalysis of transpeptidation. Rat
GGT (and presumably HuGGT) has strict stereospecificity for l-amino acid acceptor substrates, while CapD can utilize both l- and d-acceptor substrates comparably. Modeling and
kinetic analysis suggest that R520 and R432 allow two alternate acceptor
substrate binding modes for l- and d-acceptors.
R432 is conserved in <i>Francisella tularensis</i>, <i>Yersinia pestis</i>, <i>Burkholderia mallei</i>, <i>Helicobacter pylori</i> and <i>Escherichia coli</i>, but not in human GGT. Docking and MD simulations point toward key
residues that contribute to inhibitor and acceptor substrate binding,
providing a guide to designing novel and specific GGT inhibitors
Fungal bis-Naphthopyrones as Inhibitors of Botulinum Neurotoxin Serotype A
An in silico screen of the NIH Molecular Library Small
Molecule
Repository (MLSMR) of ∼350000 compounds and confirmatory bioassays
led to identification of chaetochromin A (<b>1</b>) as an inhibitor
of botulinum neurotoxin serotype A (BoNT A). Subsequent acquisition
and testing of analogues of <b>1</b> uncovered two compounds,
talaroderxines A (<b>2</b>) and B (<b>3</b>), with improved
activity. These are the first fungal metabolites reported to exhibit
BoNT/A inhibitory activity