Alanine dehydrogenase and its applications - A review.

Alanine dehydrogenase (AlaDH) (E.C.1.4.1.1) is a microbial enzyme that catalyzes a reversible conversion of L-alanine to pyruvate. Inter-conversion of alanine and pyruvate by AlaDH is central to metabolism in microorganisms. Its oxidative deamination reaction produces pyruvate which plays a pivotal role in the generation of energy through the tricarboxylic acid cycle for sporulation in the microorganisms. Its reductive amination reaction provides a route for the incorporation of ammonia and produces L-alanine which is required for synthesis of the peptidoglycan layer, proteins, and other amino acids. Also, AlaDH helps in redox balancing as its deamination/amination reaction is linked to the reduction/oxidation of NAD+/NADH in microorganisms. AlaDH from a few microorganisms can also reduce glyoxylate into glycine (aminoacetate) in a nonreversible reaction. Both its oxidative and reductive reactions exhibit remarkable applications in the pharmaceutical, environmental, and food industries. The literature addressing the characteristics and applications of AlaDH from a wide range of microorganisms is summarized in the current review.

Improvement of bilirubin oxidase productivity of Myrothecium verrucaria and studies on the enzyme overproduced by the mutant strain in the solid-state fermentation.

Bilirubin oxidase has applications in the health and environmental sectors. Hence, several attempts have been made to increase enzyme yields. However, improvements were not very high. We report here the development of a mutant strain of Myrothecium verrucaria by using UV-rays, which produced 28.8 times more enzyme compared with the parent and was higher than the yields reported in earlier submerged cultures. The mutant strain produced 35.6 times more enzyme than the parent in solid-state fermentation, which is better than that previously reported for a solid-state fermentation process. The specific activity of the enzyme produced by the mutant was higher than that of the parental enzyme. Bilirubin oxidase from both strains showed an optimum activity at pH 7 and 40°C. However, the time required to inactivate half of the initial enzyme activity at 60°C was much higher in the case of the enzyme obtained from the mutant compared with the parental enzyme. The improved thermostability of the enzyme from the mutant strain could be due to the point mutations induced during the UV irradiation, since there was no change in the mass of the enzyme compared with the parental enzyme. The bilirubin oxidase of the mutant strain degraded the bilirubin faster than the enzyme obtained from the parent under similar conditions. Faster activity of the enzyme obtained from the mutant strain could be due to its lower Km (79.4 µM) compared with that of the parental enzyme (184 µM). Hence, the mutant enzyme showed a better functionality and thermostability, which will be beneficial for industrial applications.

The 46 kDa dimeric protein from Variovorax paradoxus shows faster methotrexate degrading activity in its nanoform compare to the native enzyme.

Methotrexate degrading enzymes are required to overcome the toxicity of the methotrexate while treating the cancer. The enzyme from Variovorax paradoxus converts the methotrexate in to non toxic products. Methotrexate degrading enzyme from V. paradoxus is a dimeric protein with a molecular mass of 46 kDa and it acts on casein and gelatin. This enzyme is optimally active at pH 7.5 and 40°C and nanoparticles of this enzyme were prepared by desolvation-crosslinking method. Enzyme nanoparticles could degrade methotrexate faster than the native enzyme and they show lower Km compare to the native enzyme. Enzyme nanoparticles show better thermostability and they were stable for much longer time in the serum compare to the native enzyme. Enzyme nanoparticles show better functionality than the native enzyme while clearing the methotrexate added to the serum suggesting their advantage over the native enzyme for the therapeutic and biotechnological applications.

Development of a bioautographic method for the detection of lipase inhibitors.

An autobiographic method based on the thin layer chromatogram was developed by using the chemical system that comprises p-Nitrophenyl butyrate and bromothymol blue for detecting the lipase inhibitor. Lipase inhibitory zones were visualized as blue spots against the greenish yellow background. This method could able to detect the well known lipase inhibitor, orlistat up to the concentration of 1ng which is better than the earlier method. This method could also able to detect the lipase inhibition activities from the un-explored species of Streptomyces. The developed method can be used not only for the screening of unknown samples for the lipase inhibitors but also for the purification of the lipase inhibitors from the unknown samples.

HSV-2 inhibits type-I interferon signaling via multiple complementary and compensatory STAT2-associated mechanisms.

Type-I interferon (IFN)-mediated responses are a crucial first line of defense against viral infections and are critical for generating both innate and adaptive immunity. Therefore, viruses have necessarily evolved mechanisms to impede the IFN response. HSV-2 was found to completely abolish type-1 IFN-mediated signaling via multiple STAT2-associated mechanisms. Although the extent and kinetics of this inactivation were indistinguishable between the various cell-lines examined, there were distinct differences in the mechanisms HSV-2 employed to subvert IFN-signaling among the cell-lines. These mechanistic differences could be segregated into two categories dependent on the phase of the HSV replicative cycle that was responsible for this inhibition: (1) early phase-inhibited cells which exhibited abrogation of IFN-signaling prior to viral DNA replication; (2) late phase-inhibited cells where early phase inhibition mechanisms were not functional, but viral functions expressed following DNA replication compensated for their ineffectiveness. In early phase-inhibited cells, HSV-2 infection targeted STAT2 protein for proteosomal degradation and prevented de novo expression of STAT2 by degrading its mRNA. In contrast, HSV-2 infected late phase-inhibited cells exhibited no apparent changes in STAT2 transcript or protein levels. However, in these cells STAT2 was not activated by phosphorylation and failed to translocate to the cell nucleus, thereby preventing transactivation of antiviral genes. In primary human fibroblasts, HSV-2 failed to fully degrade STAT2 and therefore, both early and late phase mechanisms functioned cooperatively to subvert IFN-mediated antiviral gene expression. Taken together, these results indicate the importance that HSV-2 has assigned to STAT2, investing significant genomic currency throughout its replicative lifecycle for continuous targeted destruction and inhibition of this protein.

c-Myc-induced aberrant DNA synthesis and activation of DNA damage response in p300 knockdown cells.

We previously showed that in quiescent cells, p300/CBP (CREB-binding protein)family coactivators repress c-myc and prevent premature induction of DNA synthesis. p300/CBP-depleted cells exit G(1) early and continue to accumulate in S phase but do not progress into G(2)/M, and eventually they die of apoptosis. Here, we show that the S-phase arrest in these cells is because of an intra-S-phase block. The inappropriate DNA synthesis that occurs as a result of forced expression of c-myc leads to the activation of the DNA damage response as evidenced by the phosphorylation of several checkpoint related proteins and the formation of foci containing gamma-H2AX. The activation of checkpoint response is related to the induction of c-myc, as the phosphorylation of checkpoint proteins can be reversed when cells are treated with a c-Myc inhibitor or when Myc synthesis is blocked by short hairpin RNA. Using the DNA fiber assay, we show that in p300-depleted cells initiation of replication occurs from multiple replication origins. Chromatin loading of the Cdc45 protein also indicates increased origin activity in p300 knockdown cells. Immunofluorescence experiments indicate that c-Myc colocalizes with replication foci, consistent with the recently reported direct role of c-Myc in the initiation of DNA synthesis. Thus, the inappropriate S-phase entry of p300 down-regulated cells is likely to be because of c-Myc-induced deregulated replication origin activity, which results in replicative stress, activation of a DNA damage response, and S-phase arrest. Our results point to an important role for p300 in maintaining genomic integrity by negatively regulating c-myc.

Adenovirus transforming protein E1A induces c-Myc in quiescent cells by a novel mechanism.

Previously we showed that the E1A binding proteins p300 and CBP negatively regulate c-Myc in quiescent cells and that binding of E1A to p300 results in the induction of c-Myc and thereby induction of S phase. We demonstrated that p300 and HDAC3 cooperate with the transcription factor YY1 at an upstream YY1 binding site and repress the Myc promoter. Here we show that the small E1A protein induces c-Myc by interfering with the protein-protein interaction between p300, YY1, and HDAC3. Wild-type E1A but not the E1A mutants that do not bind to p300 interfered in recruitment of YY1, p300, and HDAC3 to the YY1 binding site. As E1A started to accumulate after infection, it transiently associated with promoter-bound p300. Subsequently, YY1, p300, and HDAC3 began to dissociate from the promoter. Later in infection, E1A dissociated from the promoter as well as p300, YY1, and HDAC3. Removal of HDAC3 from the promoter correlated with increased acetylation of Myc chromatin and induction. In vivo E1A stably associated with p300 and dissociated YY1 and HDAC3 from the trimolecular complex. In vitro protein-protein interaction studies indicated that E1A initially binds to the p300-YY1-HDAC3 complex, briefly associates with it, and then dissociates the complex, recapitulating somewhat the in vivo situation. Thus, E1A binding to the C-terminal region of p300 disrupts the important corepressor function provided by p300 in repressing c-Myc. Our results reveal a novel mechanism by which a viral oncoprotein activates c-Myc in quiescent cells and raise the possibility that the oncoproteins encoded by the small-DNA tumor viruses may use this mechanism to induce c-Myc, which may be critical for cell transformation.

Simian virus 40 large T overcomes p300 repression of c-Myc.

We previously showed that in quiescent cells p300/CBP negatively regulates the cell cycle G1-S transition by keeping c-Myc in a repressed state and that adenovirus E1A induces c-Myc by binding to p300/CBP. Studies have shown that p300/CBP binding to simian virus 40 large T is indirect and mediated by p53. By using a series of large T mutants that fail to bind to various cellular proteins including p53 as well as cells where p300 is overexpressed or p53 is knocked down, we show that the association of large T with p300 contributes to the induction of c-Myc and the cell cycle. The induction of c-Myc by this mechanism is likely to be important in large T mediated cell cycle induction and cell transformation.

Partially saturated canthaxanthin purified from Aspergillus carbonarius induces apoptosis in prostrate cancer cell line.

A mutant Aspergillus carbonarius selected for temperature tolerance after UV treatment, when grown in shake flasks, produced mycelia bearing yellow pigment. Since the mutant was affected in sterol biosynthetic pathway, the pigment was apparently produced to maintain membrane fluidity and rigidity for growth sustenance in low-pH culture broth. Nuclear magnetic resonance analyses characterizing the pigment as a partially saturated canthaxanthin, containing beta-ionone end rings, suggested its application as a retinoid. When tested for this property in retinoic acid receptor expressing prostate cancer cell line, LNCaP, the fungal partially saturated canthaxanthin induced apoptosis. Low apoptosis percentage in DU145 prostrate cancer cells that does not express functional retinoic acid receptor-beta (RAR-beta) suggested binding specificity of the partially saturated canthaxanthin for RAR-beta.