4 research outputs found
Systematic Functional Analysis of Active-Site Residues in l‑Threonine Dehydrogenase from Thermoplasma volcanium
Enzymes have been through millions
of years of evolution during
which their active-site microenvironments are fine-tuned. Active-site
residues are commonly conserved within protein families, indicating
their importance for substrate recognition and catalysis. In this
work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs.
Our results demonstrate that only a subset of these residues plays
an essential role in substrate recognition and catalysis and that
the native enzyme activity can be further enhanced roughly 4.6-fold
by a single point mutation. Kinetic characterization of mutants on
substrate analogs shows that l-threonine dehydrogenase possesses
promiscuous activities toward other chemically similar compounds not
previously observed. Quantum chemical calculations on the hydride-donating
ability of these substrates also reveal that this enzyme did not evolve
to harness the intrinsic substrate reactivity for enzyme catalysis.
Our analysis provides insights into connections between the details
of enzyme active-site structure and specific function. These results
are directly applicable to rational enzyme design and engineering
Comparison of Small Molecule Inhibitors of the Bacterial Cell Division Protein FtsZ and Identification of a Reliable Cross-Species Inhibitor
FtsZ is a guanosine triphosphatase (GTPase) that mediates
cytokinesis
in bacteria. FtsZ is homologous in structure to eukaryotic tubulin
and polymerizes in a similar head-to-tail fashion. The study of tubulin’s
function in eukaryotic cells has benefited greatly from specific and
potent small molecule inhibitors, including colchicine and taxol.
Although many small molecule inhibitors of FtsZ have been reported,
none has emerged as a generally useful probe for modulating bacterial
cell division. With the goal of establishing a useful and reliable
small molecule inhibitor of FtsZ, a broad biochemical cross-comparison
of reported FtsZ inhibitors was undertaken. Several of these molecules,
including phenolic natural products, are unselective inhibitors that
seem to derive their activity from the formation of microscopic colloids
or aggregates. Other compounds, including the natural product viriditoxin
and the drug development candidate PC190723, exhibit no inhibition
of GTPase activity using protocols in this work or under published
conditions. Of the compounds studied, only zantrin Z3 exhibits good
levels of inhibition, maintains activity under conditions that disrupt
small molecule aggregates, and provides a platform for exploration
of structure–activity relationships (SAR). Preliminary SAR
studies have identified slight modifications to the two side chains
of this structure that modulate the inhibitory activity of zantrin
Z3. Collectively, these studies will help focus future investigations
toward the establishment of probes for FtsZ that fill the roles of
colchicine and taxol in studies of tubulin
Elucidating Substrate Promiscuity within the FabI Enzyme Family
The rapidly growing
appreciation of enzymes’ catalytic and
substrate promiscuity may lead to their expanded use in the fields
of chemical synthesis and industrial biotechnology. Here, we explore
the substrate promiscuity of enoyl-acyl carrier protein reductases
(commonly known as FabI) and how that promiscuity is a function of
inherent reactivity and the geometric demands of the enzyme’s
active site. We demonstrate that these enzymes catalyze the reduction
of a wide range of substrates, particularly α,β-unsaturated
aldehydes. In addition, we demonstrate that a combination of quantum
mechanical hydride affinity calculations and molecular docking can
be used to rapidly categorize compounds that FabI can use as substrates.
The results here provide new insight into the determinants of catalysis
for FabI and set the stage for the development of a new assay for
drug discovery, organic synthesis, and novel biocatalysts
Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ
The
bacterial cell division protein FtsZ is one of many potential
targets for the development of novel antibiotics. Recently, zantrin
Z3 was shown to be a cross-species inhibitor of FtsZ; however, its
specific interactions with the protein are still unknown. Herein we
report the synthesis of analogues that contain a more tractable core
structure and an analogue with single-digit micromolar inhibition
of FtsZ’s GTPase activity, which represents the most potent
inhibitor of <i>Escherichia coli</i> FtsZ reported to date.
In addition, the zantrin Z3 core has been converted to two potential
photo-cross-linking reagents for proteomic studies that could shed
light on the molecular interactions between FtsZ and molecules related
to zantrin Z3