13 research outputs found
Biophysical Characterization of the vOTU Proteases from the CCHF and Dugbe Nairoviruses
My research focuses on understanding the substrate specificity of the viral homolog of the ovarian tumor domain protease superfamily (vOTU) from nairoviruses, and the structural reasons for their specificities. The vOTU from the Crimean-Congo Hemorrhagic Fever Virus (CCHFV) has been implicated in the down-regulation of the innate immune response through its ability to cleave post-translational modifications via Ubiquitin (Ub) as well as the Ub-like interferon-stimulated gene 15 (ISG15). vOTU homologs have been found in numerous viruses across several families. Moreover, the effects of these viruses range in severity from mild flu-like symptoms to mortality depending on the species of the infected host. As such, several nairovirus vOTUs including those from the Dugbe Virus (DUGV), Erve Virus (ERVEV), and CCHFV were subjected to enzymological studies to gain insight into substrate specificity. These studies revealed that even vOTUs from the same viral family have differing specificities for Ub and ISG15. Furthermore, these preferences extend to include the different polymeric moieties of Ub. In order to gain insight into any structural reasoning for these substrate predilections, the X-ray crystal structures of the vOTUs from both CCHFV and DUGV were each solved covalently bound with Ub. These structures exposed unique secondary structure elements compared to other members of the OTU superfamily that offer understanding into why certain vOTUs, such as that from CCHFV, can possess robust activity for Ub and ISG15. Likewise, the crystallographic data point to the primary structure of the vOTUs as the main driving force for divergence between nairovirus vOTU specificity
A oncompetitive inhibitor for mycobacterium tuberculosis \u27s class iia fructose 1,6-bisphosphate Aldolase
Class II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis. Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II FBAs. As such, class II FBA is considered a putative pharmacological target for the development of novel antibiotics against pathogenic bacteria such as Mycobacterium tuberculosis, the causative agent for tuberculosis (TB). To date, several competitive class II FBA substrate mimic-styled inhibitors have been developed; however, they lack either specificity, potency, or properties that limit their potential as possible therapeutics. Recently, through the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA) that has an IC50 of 10 ± 1 μM for the class II FBA present in M. tuberculosis (MtFBA). As opposed to previous inhibitors, HCA behaves in a noncompetitive manner, shows no inhibitory properties toward human and rabbit class I FBAs, and possesses anti-TB properties. Furthermore, we were able to determine the crystal structure of HCA bound to MtFBA to 2.1 Å. HCA also demonstrates inhibitory effects for other class II FBAs, including pathogenic bacteria such as methicillin-resistant Staphylococcus aureus. With its broad-spectrum potential, unique inhibitory characteristics, and flexibility of functionalization, the HCA scaffold likely represents an important advancement in the development of class II FBA inhibitors that can serve as viable preclinical candidates. © 2013 American Chemical Society
A Noncompetitive Inhibitor for <i>Mycobacterium tuberculosis</i>’s Class IIa Fructose 1,6-Bisphosphate Aldolase
Class
II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme
critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis.
Importantly, humans lack this type of aldolase, having instead a class
I FBA that is structurally and mechanistically distinct from class
II FBAs. As such, class II FBA is considered a putative pharmacological
target for the development of novel antibiotics against pathogenic
bacteria such as <i>Mycobacterium tuberculosis</i>, the
causative agent for tuberculosis (TB). To date, several competitive
class II FBA substrate mimic-styled inhibitors have been developed;
however, they lack either specificity, potency, or properties that
limit their potential as possible therapeutics. Recently, through
the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA)
that has an IC<sub>50</sub> of 10 ± 1 μM for the class
II FBA present in <i>M. tuberculosis</i> (MtFBA). As opposed
to previous inhibitors, HCA behaves in a noncompetitive manner, shows
no inhibitory properties toward human and rabbit class I FBAs, and
possesses anti-TB properties.
Furthermore, we were able to determine the crystal structure of HCA
bound to MtFBA to 2.1 Ă…. HCA also demonstrates inhibitory effects
for other class II FBAs,
including pathogenic bacteria such as methicillin-resistant <i>Staphylococcus aureus</i>. With its broad-spectrum potential,
unique inhibitory characteristics,
and flexibility of functionalization, the HCA scaffold likely represents
an important advancement in the development of class II FBA inhibitors
that can serve as viable preclinical candidates
Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from <i>Mycobacterium tuberculosis</i>
Class II fructose 1,6-bisphosphate aldolases (FBAs, EC
4.1.2.13)
comprise one of two families of aldolases. Instead of forming a Schiff
base intermediate using an ε-amino group of a lysine side chain,
class II FBAs utilize ZnÂ(II) to stabilize a proposed hydroxyenolate
intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate,
forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate
(DHAP). As class II FBAs have been shown to be essential in pathogenic
bacteria, focus has been placed on these enzymes as potential antibacterial
targets. Although structural studies of class II FBAs from <i>Mycobacterium tuberculosis</i> (MtFBA), other bacteria, and
protozoa have been reported, the structure of the active site loop
responsible for catalyzing the protonation–deprotonation steps
of the reaction for class II FBAs has not yet been observed. We therefore
utilized the potent class II FBA inhibitor phosphoglycolohydroxamate
(PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and
determined the X-ray structure of the MtFBA–PGH complex to
1.58 Ă…. Remarkably, we are able to observe well-defined electron
density for the previously elusive active site loop of MtFBA trapped
in a catalytically competent orientation. Utilization of this structural
information and site-directed mutagenesis and kinetic studies conducted
on a series of residues within the active site loop revealed that
E169 facilitates a water-mediated deprotonation–protonation
step of the MtFBA reaction mechanism. Also, solvent isotope effects
on MtFBA and catalytically relevant mutants were used to probe the
effect of loop flexibility on catalytic efficiency. Additionally,
we also reveal the structure of MtFBA in its holoenzyme form
Structural and Biochemical Characterization of Human Adenylosuccinate Lyase (ADSL) and the R303C ADSL Deficiency-Associated Mutation
Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal
recessive
disorder, which causes a defect in purine metabolism resulting in
neurological and physiological symptoms. ADSL executes two nonsequential
steps in the de novo synthesis of AMP: the conversion of phosphoribosylsuccinyl-aminoimidazole
carboxamide (SAICAR) to phosphoribosylaminoimidazole carboxamide,
which occurs in the de novo synthesis of IMP, and the conversion of
adenylosuccinate to AMP, which occurs in the de novo synthesis of
AMP and also in the purine nucleotide cycle, using the same active
site. Mutation of ADSL’s arginine 303 to a cysteine is known
to lead to ADSL deficiency. Interestingly, unlike other mutations
leading to ADSL deficiency, the R303C mutation has been suggested
to more significantly affect the enzyme’s ability to catalyze
the conversion of succinyladenosine monophosphate than that of SAICAR
to their respective products. To better understand the causation of
disease due to the R303C mutation, as well as to gain insights into
why the R303C mutation potentially has a disproportional decrease
in activity toward its substrates, the wild type (WT) and the R303C
mutant of ADSL were investigated enzymatically and thermodynamically.
Additionally, the X-ray structures of ADSL in its apo form as well
as with the R303C mutation were elucidated, providing insight into
ADSL’s cooperativity. By utilizing this information, a model
for the interaction between ADSL and SAICAR is proposed