Crystal Structure of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

There are at least 250 enzymes in Mycobacterium tuberculosis (M. tuberculosis) involved in lipid metabolism. Some of the enzymes are required for bacterial survival and full virulence. The esterase Rv0045c shares little amino acid sequence similarity with other members of the esterase/lipase family. Here, we report the 3D structure of Rv0045c. Our studies demonstrated that Rv0045c is a novel member of α/β hydrolase fold family. The structure of esterase Rv0045c contains two distinct domains: the α/β fold domain and the cap domain. The active site of esterase Rv0045c is highly conserved and comprised of two residues: Ser154 and His309. We proposed that Rv0045c probably employs two kinds of enzymatic mechanisms when hydrolyzing C-O ester bonds within substrates. The structure provides insight into the hydrolysis mechanism of the C-O ester bond, and will be helpful in understanding the ester/lipid metabolism in M. tuberculosis

A simple, rapid and sensitive FRET assay for botulinum neurotoxin serotype B detection.

Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63-85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection

LC/MS analysis of specific cleavage of FVP-B by LC/B.

<p>Mixture sample of FVP-B (8.4 µM) and LC/B (200 nM) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114124#pone-0114124-g002" target="_blank">Figure 2</a> was analysed by LC/MS to detect the full length FVP-B (A), CT-product (B) and NT-product (C).</p

Spectral properties of EDANS-Dabcyl pair [45] and the flowchart of the experimental design.

<p>(A) EDANS-Dabcyl is a widely used donor-quencher pair. The optimal absorbance and emission wavelengths of EDANS are λ_abs = 336 nm and λ_em = 490 nm respectively, and for Dabcyl, the maximum absorbance wavelength is λ_abs = 472 nm, which, to a large extent, overlap with the emission spectra of EDANS. When they are in a close proximity (10–100 Å), the energy emitted from EDANS will be quenched by Dabcyl, resulting in low or no fluorescence; when they are separated upon substrate cleavage, for example in this design, the fluorescence will increase. Hence from the fluorescence intensity change, the enzyme could be detected continuously and directly. (B) Based on the principle of FRET and our previous study, we chose the optical LC/B cleavage length of VAMP2 (63–85) as the linker between EDANS-Dabcyl.</p

Limit of detection of the developed LC/B detection system.

<p>Serial LC/B concentrations were incubated with 8.4 µM FVP-B in 500 µl reaction volume for 1 h incubation at 37°C. The fluorescence intensities were potted versus the concentration of LC/B. For each concentration, at least five replicates were carried out. LOD  = 3*S/k (S means standard deviation of negative control, k means slope).</p

The specificity of the synthesized FVP-B.

<p>LCs (200 nM each) were mixed with 8.4 µM FVP-B in reaction buffer to a final volume of 500 µl, incubated at 37°C for 1 h, fluorescent intensity was measured by a Fluorescence Spectrometer in quartz cuvette. The data obtained were processed with GraphPad Prism.</p

The inhibitory effect of EDTA on LC/B.

<p>228 nM of LC/B was mixed with 8.4 µM FVP-B with reaction buffer to a final volume of 500 µl, incubated at 37°C for 1 h, and then the fluorescent intensity was measured by a Fluorescence Spectrometer in quartz cuvette (A); 100 µl reaction volume, which contained 228 nM LC/B with 8.4 µM FVP-B, were carried out in 96 well plate after 1 h incubation at 37°C (B). For simplicity, the fluorescent intensity peak at 502 nm was selected. The data obtained were generated from at least three times repeats, and then processed with Excel and GraphPad Prism, with the negative control data subtracted.</p

Optimization of the developed assay system.

<p>To optimize the assay and test the sensitivity of our detection assay, ten-fold dilution of LC/B was performed with the concentration of FVP-B fixed at 8.4 µM (A); five-fold dilution of FVP-B, with the LC/B concentration fixed at 22.8 nM (B); the fluorescent intensity change at 502 nm of further diluted LC/B to 22.8 pM with 1.68 µM FVP-B and extended incubation time (C); and a spectrum representative of 22.8 pM LC/B incubated with 1.68 µM FVP-B for extended time span (D). Viewing from the data, the detection sensitivity of the developed assay is about 22.8 nM of LC/B within 30 min, but the sensitivity can be improved to 22.8 pM of LC/B with 2 h incubation. The data were processed by Origin85 and Excel.</p