Polyphenolic acetates: A newer anti-Mycobacterial therapeutic option

The objective of our research project was screening of various highly specific substrates of Acetoxy Drug: Protein Transacytylase (M.TAase) for antimycobacterial activity. Mycobacterial culture was done in Middlebrook’s 7H9 media. Protein purification (Mycobacterial Tranacetylase, M.TAase) was done by ion exchange chromatography and its demonstration was done on SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Middlebrook’s 7H9 broth was procured from Becton Dickinson. CM-Sepharose, DEAE-Sepharose and Q-Sephharose were purchased from Amersham Pharmacia. Anti acetyl lysine polyclonal antibody was purchased from Cell Signaling. The Middlebrook 7H9 medium was used for M. smegmatis culture. The media was prepared according to the manufacturer’s instructions. The various Polyphenol acetate compounds were tested for their antimycobacterial activities. Minimal inhibitory concentrations (MIC) were calculated by Alamar blue dye assay method. The GST protein was used as a receptor protein and purified Mycobacterial Glutamine Synthetase (GS) as TAase for acetylation by DAMC. To demonstrate the TAase catalyzed acetylation of GST by DAMC, purified M.TAase (GS) was preincubated with GST and DAMC followed by western blot using anti acetyl lysine antibody, which avidly react with the acetylated proteins. The growth pattern of M. smegmatis was diminished under the influence of various polyphenolic acetates (PA) tested for their anti-mycobacterial activity. DAMC and DAMC-5-carboxylic acid was found to have MIC of 40μg/ml whereas DAMC-6-carboxylic acid was reported to have MIC value of 35μg/ml and for ellagic acid tetra acetate (EATA) it was 60μg/ml. Previous work in our lab has led to discovery of a novel enzyme acetoxy drug: protein transacetylase (TAase), catalyzing transfer of acetyl group from various polyphenolic peracetate (PA) to certain receptor proteins such as cytochromes P-450, NADPH cytochrome reductase, nitric oxide synthase (NOS) has been established in various eukaryotic as well as prokaryotic sources. PA(s) irreversible inhibitors of mammalian CYP linked MFO, possibly due to modification of cytochrome p- 450 by acetylation in a reaction catalysed by M.TAase (GS) utilizing PA(s) as a donor of acetyl groups. Accordingly, it was hypothesized that the CYP51 of mycobacteria involved in the cell wall sterol synthesis could possibly be modified by our PA(s) through the novel unknown action of GS as transacetylase leading to the death of mycobacterial cell by way of acetylation catalyzed by acetoxy drug: protein transacetylase (M.TAase or GS).Keywords: Transacetylase; Glutamine synthetase; Mycobacterium smegmatis; Polyphenolic acetates; Acetoxy drug: protein transacetylas

Differential Effects of Leptin on the Invasive Potential of Androgen-Dependent and -Independent Prostate Carcinoma Cells

Obesity has been linked with an increased risk of prostate cancer. The formation of toxic free oxygen radicals has been implicated in obesity mediated disease processes. Leptin is one of the major cytokines produced by adipocytes and controls body weight homeostasis through food intake and energy expenditure. The rationale of the study was to determine the impact of leptin on the metastatic potential of androgen-sensitive (LNCaP) cells as well as androgen-insensitive (PC-3 and DU-145) cells. At a concentration of 200 nm, LNCaP cells showed a significant increase (20% above control; P < .0001) in cellular proliferation without any effect on androgen-insensitive cells. Furthermore, exposure to leptin caused a significant (P < .01 to P < .0001) dose-dependent decrease in migration and invasion of PC3 and Du-145 prostate carcinoma cell lines. At the molecular level, exposure of androgen-independent prostate cancer cells to leptin stimulates the phosphorylation of MAPK at early time point as well as the transcription factor STAT3, suggesting the activation of the intracellular signaling cascade upon leptin binding to its cognate receptor. Taken together, these results suggest that leptin mediates the invasive potential of prostate carcinoma cells, and that this effect is dependent on their androgen sensitivity

Structural insights into the production of 3-hydroxypropionic acid by aldehyde dehydrogenase from Azospirillum brasilense

3-Hydroxypropionic acid (3-HP) is an important platform chemical to be converted to acrylic acid and acrylamide. Aldehyde dehydrogenase (ALDH), an enzyme that catalyzes the reaction of 3-hydroxypropionaldehyde (3-HPA) to 3-HP, determines 3-HP production rate during the conversion of glycerol to 3-HP. To elucidate molecular mechanism of 3-HP production, we determined the first crystal structure of a 3-HP producing ALDH, alpha-ketoglutarate-semialdehyde dehydrogenase from Azospirillum basilensis (AbKGSADH), in its apo-form and in complex with NAD(+). Although showing an overall structure similar to other ALDHs, the AbKGSADH enzyme had an optimal substrate binding site for accepting 3-HPA as a substrate. Molecular docking simulation of 3-HPA into the AbKGSADH structure revealed that the residues Asn159, Gln160 and Arg163 stabilize the aldehyde-and the hydroxyl-groups of 3-HPA through hydrogen bonds, and several hydrophobic residues, such as Phe156, Val286, Ile288, and Phe450, provide the optimal size and shape for 3-HPA binding. We also compared AbKGSADH with other reported 3-HP producing ALDHs for the crucial amino acid residues for enzyme catalysis and substrate binding, which provides structural implications on how these enzymes utilize 3-HPA as a substrate

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans . Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75×105 M−1 s−1 at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45–55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host’s transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35±0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.Web of Scienc

Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD(+), for enhancing the production of 3-hydroxypropionic acid

3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD(+) regeneration. We engineered a-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD(+), were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD(+), less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM)

The p12 Domain Is Unstructured in a Murine Leukemia Virus p12-CAN Gag Construct

The Gag polyproteins of gammaretroviruses contain a conserved p12 domain between MA and CA that plays critical roles in virus assembly, reverse transcription and nuclear integration. Here we show using nuclear magnetic resonance, that p12 is unstructured in a Moloney murine leukemia virus (MMLV) Gag fragment that includes the N-terminal domain of CA (p12-CAN). Furthermore, no long range interactions were observed between the domains, as has been previously predicted. Flexibility appears to be a common feature of Gag “late” domains required for virus release during budding. Residues near the N-terminus of CAN that form a β-hairpin in the mature CA protein are unfolded in p12-CAN, consistent with proposals that hairpin formation helps trigger capsid assembly

Expression and localisation of Akt-1, Akt-2 and Akt-3 correlate with clinical outcome of prostate cancer patients

We investigated the correlation between the expression and localisation of Akt-1, Akt-2, Akt-3, phospho-Akt proteins and the clinicopathological parameters in 63 prostate cancer specimens. More than 60% of cancerous tissues overexpressed Akt-1, Akt-2 or Akt-3. Cytoplasmic Akt-1 expression was correlated with a higher risk of postoperative prostate-specific antigen (PSA) recurrence and shorter PSA recurrence interval. Cytoplasmic Akt-2 did not show any significant correlation with clinicopathological parameters predicting outcomes. Cytoplasmic Akt-3 was associated with hormone-refractory disease progression and extracapsular invasion. Nuclear Akt-1 and Akt-2 expression were correlated with favourable outcome parameters such as absence of lymph node and perineural invasion. Kaplan–Meier analysis and Cox regression model also showed that Akt-1 and Akt-2, but not Akt-3 or phospho-Akt was associated with a significantly higher risk of PSA recurrence. In contrast, nuclear Akt-1 was significantly associated with a lower risk of PSA recurrence. Multivariate analysis revealed that clinical stage, Gleason score and the combined cytoplasmic nuclear Akt-1 marker in cancerous tissues were significant independent prognostic factors of PSA recurrence. This is the first report demonstrating in patients with prostate cancer and the particular role of Akt-1 isoform expression as a prognostic marker depending of its localisation