Molecular Mechanism for Attractant Signaling to DHMA by E. coli Tsr.

The attractant chemotaxis response of Escherichia coli to norepinephrine requires that it be converted to 3,4-dihydroxymandelic acid (DHMA) by the monoamine oxidase TynA and the aromatic aldehyde dehydrogenase FeaB. DHMA is sensed by the serine chemoreceptor Tsr, and the attractant response requires that at least one subunit of the periplasmic domain of the Tsr homodimer (pTsr) has an intact serine-binding site. DHMA that is generated in vivo by E.

A Static Microfluidic Device for Investigating the Chemotaxis Response to Stable, Non-linear Gradients.

Microfluidic technology allows fast and precise measurement of chemotaxis responses to both attractant and repellent signals. One of the major drawbacks of current microfluidic chemotaxis assays is the presence of bacterial cells within the concentration gradient flow field, which has the potential for flow effects masking the chemotaxis response. This chapter describes a new microfluidic device for producing stable concentration gradients and measuring the response of cells to the gradient without exposing them to any flow.

Conversion of Norepinephrine to 3,4-Dihdroxymandelic Acid in Escherichia coli Requires the QseBC Quorum-Sensing System and the FeaR Transcription Factor.

The detection of norepinephrine (NE) as a chemoattractant by strain K-12 requires the combined action of the TynA monoamine oxidase and the FeaB aromatic aldehyde dehydrogenase. The role of these enzymes is to convert NE into 3,4-dihydroxymandelic acid (DHMA), which is a potent chemoattractant sensed by the Tsr chemoreceptor. These two enzymes must be induced by prior exposure to NE, and cells that are exposed to NE for the first time initially show minimal chemotaxis toward it.

The Norepinephrine Metabolite 3,4-Dihydroxymandelic Acid Is Produced by the Commensal Microbiota and Promotes Chemotaxis and Virulence Gene Expression in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic (EHEC) is a commonly occurring foodborne pathogen responsible for numerous multistate outbreaks in the United States. It is known to infect the host gastrointestinal tract, specifically, in locations associated with lymphoid tissue. These niches serve as sources of enteric neurotransmitters, such as epinephrine and norepinephrine, that are known to increase virulence in several pathogens, including enterohemorrhagic The mechanisms that allow pathogens to target these niches are poorly understood.

Bridging of a substrate between cyclodextrin and an enzyme's active site pocket triggers a unique mode of inhibition.

Background: Methionyl-7-amino-4-methylcoumarin (MetAMC) serves as a substrate for the Escherichia coli methionine aminopeptidase (MetAP) catalyzed reaction, and is routinely used for screening compounds to identify potential antibiotic agents. In pursuit of screening the enzyme's inhibitors, we observed that 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), utilized to solubilize hydrophobic inhibitors, inhibited the catalytic activity of the enzyme, and such inhibition was not solely due to sequestration of the substrate by HP-β-CD.

Methods: The mechanistic path for the HP-β-CD mediated inhibition of MetAP was probed by performing a detailed account of steady-state kinetics, ligand binding, X-ray crystallographic, and molecular modeling studies.

Chemotaxis of Escherichia coli to norepinephrine (NE) requires conversion of NE to 3,4-dihydroxymandelic acid.

Norepinephrine (NE), the primary neurotransmitter of the sympathetic nervous system, has been reported to be a chemoattractant for enterohemorrhagic Escherichia coli (EHEC). Here we show that nonpathogenic E. coli K-12 grown in the presence of 2 μM NE is also attracted to NE.

Probing the metal ion selectivity in methionine aminopeptidase via changes in the luminescence properties of the enzyme bound europium ion.

We report herein, for the first time, that Europium ion (Eu(3+)) binds to the "apo" form of Escherichia coli methionine aminopeptidase (EcMetAP), and such binding results in the activation of the enzyme as well as enhancement in the luminescence intensity of the metal ion. Due to competitive displacement of the enzyme-bound Eu(3+) by different metal ions, we could determine the binding affinities of both "activating" and "non-activating" metal ions for the enzyme via fluorescence spectroscopy. The experimental data revealed that among all metal ions, Fe(2+) exhibited the highest binding affinity for the enzyme, supporting the notion that it serves as the physiological metal ion for the enzyme.

Histone deacetylase activators: N-acetylthioureas serve as highly potent and isozyme selective activators for human histone deacetylase-8 on a fluorescent substrate.

We report, for the first time, that certain N-acetylthiourea derivatives serve as highly potent and isozyme selective activators for the recombinant form of human histone deacetylase-8 in the assay system containing Fluor-de-Lys as a fluorescent substrate. The experimental data reveals that such activating feature is manifested via decrease in the K(m) value of the enzyme's substrate and increase in the catalytic turnover rate of the enzyme.

Alternative Modes of Binding of Recombinant Human Histone Deacetylase 8 to Colloidal Gold Nanoparticles.

Histone deacetylases are intimately involved in the transcriptional regulation of genes, and they are high priority drug targets for cancer therapy. Due to prevalence of several sulfhydryl groups on the surface of histone deacetylase 8, we explored the possibility of its binding to colloidal gold nanoparticles by determining its potentials to inhibit the flocculation as well as retaining the enzyme activity. It was observed that although both these processes conformed to the binding affinity of the gold-histone deacetylase 8 conjugate as being equal to 15-20 nM, only 30% of the nanoparticle-bound enzyme exhibited the enzymatic activity.

Synthesis of barbiturate-based methionine aminopeptidase-1 inhibitors.

The syntheses of a new class of barbiturate-based inhibitors for human and Escherichia Coli methionine aminopeptidase-1 (MetAP-1) are described. Some of the synthesized inhibitors show selective inhibition of the human enzyme with high potency.