Construing recombinant ZFP160 from Aspergillus terreus as pterin deaminase enzyme.

Pterin deaminase stands as a metalloenzyme and exhibits both antitumor and anticancer activities. Therefore, this study aimed to explore the molecular function of zinc finger protein-160 (zfp160) from Aspergillus terreus with its enzyme mechanism in detail. Subsequently, preliminary molecular docking studies on zfp160 from A. terreus were done. Next, the cloning and expression of zfp160 protein were carried out. Following, protein expression was induced and purified through nickel NTA column with imidazole gradient elution. Through the Mascot search engine tool, the expressed protein of MALDI-TOF was confirmed by 32 kDa bands of SDS-PAGE. Furthermore, its enzymatic characterization and biochemical categorization were also explored. The optimum conditions for enzyme were determined to be pH 8, temperature 35°C, km 50 µm with folic acid as substrate, and Vmax of 24.16 (IU/mL). Further, in silico analysis tried to explore the interactions and binding affinity of various substrates to the modeled pterin deaminase from A. terreus. Our results revealed the binding mode of different substrate molecules with pterin deaminase using the approximate scoring functions that possibly correlate with actual experimental binding affinities. Following this, molecular dynamic simulations provided the in-depth knowledge on deciphering functional mechanisms of pterin deaminase over other drugs.

Identification and structural prediction of the unrevealed amidohydrolase enzyme: Pterin deaminase from Agrobacterium tumefaciens LBA4404.

Microbes make a remarkable contribution to the health and well-being of living beings all over the world. Interestingly, pterin deaminase is an amidohydrolase enzyme that exhibits antitumor, anticancer activities and antioxidant properties. With the existing evidence of the presence of pterin deaminase from microbial sources, an attempt was made to reveal the existence of this enzyme in the unexplored bacterium Agrobacterium tumefaciens LBA4404. After, the cells were harvested and characterized as intracellular enzymes and then partially purified through acetone precipitation. Subsequently, further purification step was carried out with an ion-exchange chromatogram (HiTrap Q FF) using the Fast-Protein Liquid Chromatography technique (FPLC). Henceforward, the approximate molecular weight of the purified pterin deaminase was determined through SDS-PAGE. Furthermore, the purified protein was identified accurately by MALDI-TOF, and the sequence was explored through a Mascot search engine. Additionally, the three-dimensional structure was predicted and then validated, as well as ligand-binding sites, and the stability of this enzyme was confirmed for the first time. Thus, the present study revealed the selected parameters showing a considerable impact on the identification and purification of pterin deaminase from A. tumefaciens LBA4404 for the first time. The enzyme specificity makes it a favorable choice as a potent anticancer agent.

Pharmacological assessment of Ru(II) complex with GidA protein- A novel topoisomerase II inhibitor towards cancer therapeutics.

The interactions of ruthenium(II) complex with Glucose inhibited division protein A (GidA protein) was studied through various spectroscopic techniques with the ultimate goal of preparing adducts with good selectivity for cancer cells. In all the cases, formation of a tight metal-protein conjugate was observed. The influence of pH, reducing agents and chelators on the formation of adduct was analysed by UV- visible spectroscopy. While there was no effect on the addition of sodium ascorbate, some alterations on some selected bands were seen on the UV-visible spectra on the addition of EDTA. The adduct was stable in the pH range of 5-8. Addition of ruthenium(II) complex effectively quenched the intrinsic fluorescence of GidA and it occurred through static quenching. The effect of ruthenium(II) complex on the conformation of GidA has been examined by analyzing CD spectrum. Though, there was some conformational changes observed in the presence of ruthenium(II) complex, α- helix in the secondary structure of GidA retained its identity. Molecular docking of ruthenium(II) complex with GidA also indicated that GidA docks through hydrophobic interaction. The stable semisynthetic complex (ruthenium(II) complex with GidA) was checked for topoisomerase II inhibition. Relaxation and decatenation assay proved topoisomerase II inhibition of semisynthetic complex.Communicated by Ramaswamy H. Sarma.

Bioconversion of lovastatin to simvastatin by Streptomyces carpaticus toward the inhibition of HMG-CoA activity.

The aim of this study was the modification of lovastatin by microbes to improve its potential. Actinobacteria exhibit staggering diversity in terms of their biosynthetic capability for specialized metabolites which has been traced back to the presence of specialized gene clusters. The objective of the study is to exploit the potential of Actinobacteria strain(s), which can biotransform lovastatin to simvastatin, which might be a more potent therapeutic agent than lovastatin. We have screened 40 Actinobacteria strains and assessed their biotransformation potential primarily through thin layer chromatography (TLC) analysis, followed by high performance thin layer chromatography and high performance liquid chromatography analysis. One strain C7 (CTL S12) has been identified as a potential Actinobacteria that favored the simvastatin biotransformation. The morphological and biochemical analysis together with 16S rRNA sequencing coupled with phylogenetic analysis confirmed the ideal strain (C7) as Streptomyces carpaticus. Successively, the purified simvastatin from S. carpaticus was characterized by liquid chromatography-mass spectrometry (LC-MS), infrared spectrometry, nuclear magnetic resonance, and HMG-CoA assay. In the LC-MS analysis, a peak at 419.24 m/z confirmed the elemental composition of simvastatin (C25 H39 O5 ). In HMG-CoA assay, the IC50 of simvastatin was 50 µg/ml, and the inhibitory potential was 1.36 times higher compared to that of lovastatin. Thus, the biotransformation of simvastatin from lovastatin by S. carpaticus is reported for the first time.

Bioprospecting lovastatin production from a novel producer Cunninghamella blakesleeana.

Beside anti-cholesterol activity, lovastatin garners worldwide attention for therapeutical application against various diseases especially cancer. A total of 36 filamentous fungi from soil samples were isolated and screened for lovastatin production by yeast growth bioassay method. C9 strain (later identified as Cunninghamella blakesleeana) was screened as potential strain of lovastatin production. Further confirmation of the compound was made using TLC, HPTLC and HPLC in which similar Rf value, densitogram peak and chromatogram peak against the standard lovastatin were observed, respectively. The purified lovastatin subjected for IR analysis showed a lactone ring peak at 1763.63 cm-1 similar to standard lovastatin. Further structural analysis including NMR and LC-MS of the purified lovastatin reassures the molecular formula and molecular weight similar to standard. In quantitative terms, C. blakesleeana, Aspergillus terreus and Aspergillus flavus produced 1.4 mg g-1 DWS, 0.83 mg g-1 DWS and 0.3 mg g-1 DWS of lovastatin, respectively, (p < 0.0001) without any optimization. Lovastatin showed significant antioxidant property with IC50: 145.9 µg mL-1 (140 µL), and the percentage of inhibition is maximum at 199.5 µg/mL which is statistically significant (p < 0.0001).

Analeptic agent from microbes upon cyanide degradation.

Microbes being the initial form of life and ubiquitous in occurrence, they adapt to the environment quickly. The microbial metabolism undergoes alteration to ensure conducive environment either by degrading the toxic substances or producing toxins to protect themselves. The presence of cyanide waste triggers the cyanide degrading enzymes in the microbes which facilitate the microbes to utilize the cyanide for its growth. To enable the degradation of cyanide, the microbes also produce the necessary cofactors and enhancers catalyzing the degradation pathways. Pterin, a cofactor of the enzyme cyanide monooxygenase catalyzing the oxidation of cyanide, is considered to be a potentially bioactive compound. Besides that, the pterins also act as cofactor for the enzymes involved in neurotransmitter metabolism. The therapeutic values of pterin as neuromodulating agent validate the necessity to pursue the commercial production of pterin. Even though chemical synthesis is possible, the non-toxic methods of pterin production need to be given greater attention in future.

Characterization of unexplored amidohydrolase enzyme-pterin deaminase.

Pterin deaminase is an amidohydrolase enzyme hydrolyzing pteridines to form lumazine derivatives and ammonia. The enzyme captured the attention of scientists as early as 1959 and had been patented for its application as an anticancer agent. It is ubiquitously present in prokaryotes and has been reported in some eukaryotes such as honey bee, silkworm and rats. The enzyme has been observed to have a spectrum of substrates with the formation of respective lumazines. The role of the substrates of the enzyme in various metabolic pathways warrants a significant role in the biological activity of both prokaryotes and eukaryotes. Even though the functions of the enzyme have been explored in prokaryotes, their niche in the eukaryotic system is not clear. There is very few information on the structural and functional properties of the enzyme. This review has been congregated to emphasize the significance of pterin deaminase and analyzes the lacunae in understanding the biological characters of the enzyme.