The self‐assembly of a dehydrophenylalanine containing dipeptide (see figure), yielding highly ordered nanotubular structures, is discussed. The tubes are longer and thinner than previously reported peptide‐based tubular structures; they are stable to boiling‐water temperatures, different pH conditions, and to a highly nonspecific protease (Proteinase K).

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k cat = 2.0 s −1 ; k cat /K m = 2.5 × 10 3 M −1 s −1 ). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn 2+ prior to induction, the purified enzyme was substantially more active for the deamination of adenine with values of k cat and k cat /K m of 200 s −1 and 5 × 10 5 M −1 s −1 , respectively. The apo-enzyme was prepared and reconstituted with Fe 2+ , Zn 2+ , or Mn 2+ . In each case, two enzyme-equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member within the deaminase sub-family of the amidohydrolase superfamily (AHS) to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe II /Fe II ]-ADE was oxidized to [Fe III /Fe III ]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe III /Fe III ]-ADE with dithionite restored the deaminase activity and thus the di-ferrous form of the enzyme is essential for catalytic activity. No evidence for spin-coupling between metal ions was evident by EPR or Mössbauer spectroscopies. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by Xray crystallography at 2.2 Å resolution and adenine was modeled into the active site based on homology to other members of the amidohydrolase superfamily. Based on the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH rate profiles and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate limiting steps. † This work was supported in part by the National Institutes of Health (GM 71790, GM074945, and GM 46441). The X-ray coordinates and structure factors for Atu4426 have been deposited in the Protein Data Bank (PDB accession code: 3nqb) * To whom correspondence may be sent: (FMR) Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 March 22.Published in final edited form as: Biochemistry. 2011 March 22; 50(11): 1917-1927. doi:10.1021/bi101788n. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAdenine deaminase (ADE 1 ) catalyzes the conversion of adenine to hypoxanthine and ammonia as shown in Scheme 1 (1,2). ADE is part of the purine degradation pathway where hypoxanthine is subsequently oxidized to uric acid by xanthine oxidase via a xanthine intermediate (3). This enzyme also participates in the purine salvage pathway for the synthesis of guanine nucleotides (1). ADE from Escherichia coli is a member of the amidohydrolase superfamily (AHS) and is clustered within cog1001 in ...

Abrin and agglutinin-I from the seeds of Abrus precatorius are type II ribosome-inactivating proteins that inhibit protein synthesis in eukaryotic cells. The two toxins share a high degree of sequence similarity; however, agglutinin-I is weaker in its activity. We compared the kinetics of protein synthesis inhibition by abrin and agglutinin-I in two different cell lines and found that ϳ200 -2000-fold higher concentration of agglutinin-I is needed for the same degree of inhibition. Like abrin, agglutinin-I also induced apoptosis in the cells by triggering the intrinsic mitochondrial pathway, although at higher concentrations as compared with abrin. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison with that of the reported structure of abrin. The overall protein folding of agglutinin-I is similar to that of abrin-a with a single disulfide bond holding the toxic A subunit and the lectin-like B-subunit together, constituting a heterodimer. However, there are significant differences in the secondary structural elements, mostly in the A chain. The substitution of Asn-200 in abrin-a with Pro-199 in agglutinin-I seems to be a major cause for the decreased toxicity of agglutinin-I. This perhaps is not a consequence of any kink formation by a proline residue in the helical segment, as reported by others earlier, but due to fewer interactions that proline can possibly have with the bound substrate.

The arid and semi-arid regions of Rajasthan are one of the most extreme biomes of India, possessing diverse microbial communities that exhibit immense biotechnological potential for industries. Herein, we sampled study sites from arid and semi-arid regions of Thar Desert, Rajasthan, India and subjected them to chemical, physical and metagenomics analysis. The microbial diversity was studied using V3–V4 amplicon sequencing of 16S rRNA gene by Illumina MiSeq. Our metagenomic analyses revealed that the sampled sites consist mainly of Proteobacteria (19–31%) followed by unclassified bacteria (5–21%), Actinobacteria (3–25%), Planctomycetes (5–13%), Chloroflexi (2–14%), Bacteroidetes (3–12%), Firmicutes (3–7%), Acidobacteria (1–4%) and Patescibacteria (1–4%). We have found Proteobacteria in abundance which is associated with a range of activities involved in biogeochemical cycles such as carbon, nitrogen, and sulphur. Our study is perhaps the first of its kind to explore soil bacteria from arid and semi-arid regions of Rajasthan, India. We believe that the new microbial candidates found can be further explored for various industrial and biotechnological applications.

The current investigation involves the green synthesis of copper nanoparticles (CuNPs) from an aqueous plant extract of Moringa oleifera Lam by two methods: (I) time-based approach and (II) heat treatment of aqueous solution. Prepared CuNPs were characterised via Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy and transmission EM. The study also reveals the potential bioactivity of the prepared CuNPs. In vitro anti-microbial efficiency of CuNPs was estimated against bacterial and fungal strains by the agar well diffusion method. Anti-oxidant capacity of CuNPs was determined using ferric reducing ability of plasma (FRAP), lipid peroxidation (LPO) and peroxidase assays, while the antiplatelet potential was determined by measuring two haemostatic parameters (PT & APTT assay). The minimum inhibitory concentration was observed at 60 µg/ml against Streptomyces griseus and Aspergillus niger when NPs were prepared by method II. CuNPs prepared by the method I showed higher FRAP and LPO activities, while increased POX activity was found in CuNPs prepared by method II. CuNPs prepared using method I also showed better anti-oxidant and antiplatelet potential. It was observed that M. oleifera-derived CuNPs exhibits strong anti-microbial, anti-oxidant and APTT potential. This indicates potential utilization of green synthesized NPs for various industrial and therapeutic strategies.

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn 21 before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO 4 . Inductively coupled plasma mass spectrometry and Mö ssbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [ ]-ADE underwent more than 100 turnovers with H 2 O 2 before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g ave 5 4.3 EPR signal and no evidence of antiferromagnetic spin-coupling. A model is proposed for the disproportionation of H 2 O 2 by [Fe II /Fe II ]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

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This study reports the instantaneous synthesis of stable silver nanoparticles (AgNPs) at room temperature using a digallate (DG) derivative linked through hydrophilic oxyethylene spacer. The DG derivative was synthesized using standard ester coupling reaction and employed for the production of AgNPs. The Digallate acted as a very strong reducing and stabilizing agent as it reduced Ag ions in an aqueous media into Ag nanoparticles (DG-AgNPs) instantaneously at room temperature. The DG-AgNPs were characterized by UV/Vis, XPS, XRD, HR-TEM, SEM, AFM, DLS and zeta potential measurements and were evaluated for their antibacterial potential against four different strains (Escherichia coli, Staphylococcus aureus, Streptomyces griseus and Bacillus subtilis). It demonstrated strong inhibitory activity with minimum inhibitory concentrations (MIC) ranging from 4.0-6.0 μg/ml against all the tested bacterial strains. Optical density (OD) measurements and live/dead cell staining assay revealed significant antibacterial action of DG-AgNPs. The MTT based cytotoxicity, cellular morphology and nuclear staining analysis of NIH 3T3 cells (fibroblasts) suggested DG-AgNPs to be relatively safe as it did not reveal any growth inhibition for the optimized antibacterial concentrations. Concisely, relatively non-toxic AgNPs have been produced rapidly at room temperature using dimeric gallate and suggested as effective and promising antibacterial agent for pharmaceutical applications.