33 research outputs found
Crystal Structure of the Cofactor-Binding Domain of the Human Phase II Drug-Metabolism Enzyme UDP-Glucuronosyltransferase 2B7
Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in the processing of a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8 â„« resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designed UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicate that human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting that human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer
Characterization of human hepatic and extrahepatic UDP-glucuronosyltransferase (UGTs) enzymes involved in the metabolism of classical cannabinoids
saeRS and sarA Act Synergistically to Repress Protease Production and Promote Biofilm Formation in Staphylococcus aureus
Mutation of the staphylococcal accessory regulator (sarA) limits biofilm formation in diverse strains of Staphylococcus aureus, but there are exceptions. One of these is the commonly studied strain Newman. This strain has two defects of potential relevance, the first being mutations that preclude anchoring of the fibronectin-binding proteins FnbA and FnbB to the cell wall, and the second being a point mutation in saeS that results in constitutive activation of the saePQRS regulatory system. We repaired these defects to determine whether either plays a role in biofilm formation and, if so, whether this could account for the reduced impact of sarA in Newman. Restoration of surface-anchored FnbA enhanced biofilm formation, but mutation of sarA in this fnbA-positive strain increased rather than decreased biofilm formation. Mutation of sarA in an saeS-repaired derivative of Newman (P18L) or a Newman saeRS mutant (ΔsaeRS) resulted in a biofilm-deficient phenotype like that observed in clinical isolates, even in the absence of surface-anchored FnbA. These phenotypes were correlated with increased production of extracellular proteases and decreased accumulation of FnbA and/or Spa in the P18L and ΔsaeRS sarA mutants by comparison to the Newman sarA mutant. The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon. These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm. Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level
Epistatic Relationships between sarA and agr in Staphylococcus aureus Biofilm Formation
Background: The accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci.
Methodology/Principal Findings: We generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to forma biofilmin the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard.
Conclusions and Significance: The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host
Identification of Hydroxywarfarin Binding Site in Human UDP Glucuronosyltransferase 1A10: Phenylalanine 90
Characterization of Human Hepatic and Extrahepatic UDP-Glucuronosyltransferase Enzymes Involved in the Metabolism of Classic Cannabinoids
Tetrahydrocannabinol (Δ9-THC), the primary psychoactive
ingredient in marijuana, is subject to cytochrome P450 oxidation and
subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many
studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible
for these cytochrome P450-dependent oxidations, but little work has been done
to characterize phase II metabolic pathways. In this study, we test the
hypothesis that there are specific human UGTs responsible for classic
cannabinoid metabolism. The activities of 12 human recombinant UGTs toward
classic cannabinoids [cannabinol (CBN), cannabidiol (CBD),
(–)-Δ8-THC, (–)-Δ9-THC,
(±)-11-hydroxy-Δ9-THC (THC-OH), and
(–)-11-nor-9-carboxy-Δ9-THC (THC-COOH)] were evaluated
using high-performance liquid chromatography-tandem mass spectrometry and
labeling assays. Despite activity by UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7
toward CBN, CBD, THC-OH, and THC-COOH, only selected UGTs demonstrate
sufficient activity for further characterization of steady-state kinetics. CBN
was the most recognized substrate as evidenced by activities from hepatic
UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect
the introduction of an aromatic ring to Δ9-THC, leading to
favorable π stacking with phenylalanines in the UGT active site. Likewise,
oxidation of Δ9-THC to THC-OH results in UGT1A9 and UGT1A10
activity toward the cannabinoid. Further oxidation to THC-COOH surprisingly
leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a
substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of
THC-COOH is the main metabolite found in urine, and thus these hepatic enzymes
play a critical role in the metabolic clearance of cannabinoids. Taken
together, glucuronidation of cannabinoids depends on upstream processing
including enzymes such as CYP2C9 and CYP3A4
The First Aspartic Acid of the DQxD Motif for Human UDP-Glucuronosyltransferase 1A10 Interacts with UDP-Glucuronic Acid during Catalysis
Characterization of Human Hepatic and Extrahepatic UDP-Glucuronosyltransferase Enzymes Involved in the Metabolism of Classic Cannabinoids
ABSTRACT: Tetrahydrocannabinol (⌬ 9 -THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UG
Impact of <i>saeRS</i> and surface-associated FnbA on biofilm formation in Newman.
<p>Surface-anchored FnbA was restored in Newman (New), its <i>saeS</i>-repaired derivative (P18L), and its isogenic <i>saeRS</i> mutant (<i>sae</i>) by introduction of a plasmid-borne copy of <i>fnbA</i>. Biofilm formation was assessed using a microtiter plate assay, with UAMS-1 (U1) and its <i>sarA</i> mutant included as positive and negative controls, respectively. <i>sarA</i> mutants are designated as “S.” Asterisks indicate statistical significance (p<0.05) by comparison to the isogenic parent strain (WT).</p