Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.     

Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme

Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for beta cell function, suggesting a vital framework for the regulation of glucose homeostasis.     

Phylogenetic Study of Methanol Oxidizers from Chilika-Lake Sediments Using Genomic and Metagenomic Approaches

Group-wise diversity of sediment methylotrophs of Chilika lake (Lat. 19°28′–19°54′N; Long. 85°06′–85°35′E) Odisha, India at various identified sites was studied. Both the culturable and unculturable (metagenome) methylotrophs were investigated in the lake sediments employing both mxaF and 16S rRNA genes as markers. ARDRA profiling, 16S rRNA gene sequencing, PAGE profiling of HaeIII, EcoRI restricted mxaF gene and the mxaF gene sequences using culture-dependent approach revealed the relatedness of α-proteobacteria and Methylobacterium, Hyphomicrobium and Ancyclobacter sp. The total viable counts of the culturable aerobic methylotrophs were relatively higher in sediments near the sea mouth (S3; Panaspada), also demonstrated relatively high salinity (0.1 M NaCl) tolerance. Metagenomic DNA from the sediments, amplified using GC clamp mxaF primers and resolved through DGGE, revealed the diversity within the unculturable methylotrophic bacterium Methylobacterium organophilum, Ancyclobacter aquaticus, Burkholderiales and Hyphomicrobium sp. Culture-independent analyses revealed that up to 90 % of the methylotrophs were unculturable. The study enhances the general understandings of the metagenomic methylotrophs from such a special ecological niche.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0510-3) contains supplementary material, which is available to authorized users.     

Crystal structure of aspartyl dipeptidase from Xenopus laevis revealed ligand binding induced loop ordering and catalytic triad assembly

Gene encoding aspartyl dipeptidase from Xenopus levies (PepExl) is upregulated by thyroid hormone and is proposed to play a significant role in resorption of tadpole tail during metamorphosis. However, the importance of peptidase activity for the resorption of the tail remain elusive. Here we report the crystal structures of first eukaryotic S51 peptidase, PepExl, in its ligand-free and Asp-bound states at 1.4 and 1.8 Å resolutions, respectively. The active site is located at dimeric interface and the catalytic triad is found to be dissembled in ligand-free and assembled in Asp-bound state. Structural comparison and molecular dynamic simulations of ligand-free and Asp-bound states shows that distinct loop (loop-A) plays an important role in active site shielding, substrate binding and enzyme activation. This study illuminates the Asp-X dipeptide binding in PepExl is associated with ordering of the loop-A and assembly of residues of catalytic triad in active conformation for enzymatic activity.     

Soral crypsis: protective mimicry of a coccid on an Indian fern

Herbivory with crypsis is not well documented in ferns. The present record of cryptic coloration of coccid Saissetia filicum Boisduval (Homoptera: Coccidae) to the sori of a fern species Asplenium nidus L. (Aspleniaceae) is unique. Predatory beetles (Jauravia sp., Coleoptera: Coccinellidae) that feed on the coccids, are suggested to be selective pressure for the development of the present homopteran soral crypsis. A higher rate of effective predation is noticed in the vegetative leaves than the fertile leaves. Aggressive ants were found harvesting honeydew secretions from the coccids and defending the trophobionts as well as the host fern from their natural enemies. In addition, a possible three-way mutualistic relationship among the coccids, its host fern and the tending ant is suggested. Differential numbers of coccids on vegetative and fertile leaves is correlated with their phenol content and degree of predation by beetles. Such coloration mimicry by the coccids may enable them to obtain the necessary blend of sorus of the host fern needed to evade beetle detection and attack.     

Overexpression and purification of recombinant eel calcitonin and its phylogenetic analysis

Calcitonin (CT), a peptide hormone that is widely used for the treatment of osteoporosis, Paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients, is produced by the para-follicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Fish calcitonin, like eel calcitonin (eCT), is more potent and longer lasting than human CT and is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated eCT in recombinant Streptomyces avermitilis. A phylogenetic analysis was performed with all the known CT amino acid sequences.     

Stabilizing Multi-Electron NASICON-Na1.5V0.5Nb1.5(PO4)3 Anode via Structural Modulation for Long-Life Sodium-Ion Batteries

Multi-electron NAtrium SuperIonic CONductor (NASICON)-Nb₂(PO₄)₃ (N₀NbP) is an attractive Na-ion battery anode, owing to its low intercalation voltage (1.4 V vs Na⁺/Na⁰) and high capacity (≈150 mAh g⁻¹). However, it suffers from poor capacity retention due to structural degradation. To overcome this issue, extra Na⁺ ions are introduced at the Na(1) sites, via V³⁺ substitution, which can act as stabilizing agents to hold lantern units together during cycling, producing NASICON-Na_1.5V_0.5Nb_1.5(PO₄)₃ (N_1.5VNbP). The N_1.5VNbP anode exhibits reversible capacities of ≈140 mAh g⁻¹ at 1.4 V versus Na⁺/Na⁰ through Nb⁵⁺/Nb⁴⁺/Nb³⁺ and V³⁺/V²⁺ redox activities. The extra Na⁺ ions in the framework forms a complete solid-solution during Na (de)intercalation and enhances sodium diffusivity, in agreement with first-principles calculations. Further, N_1.5VNbP demonstrates extraordinary cycling (89% capacity retention at 5C after 500 cycles) and rate performances (105 mAh g⁻¹ at 5C). Upon pairing the N_1.5VNbP anode with the NASICON-Na₃V₂(PO₄)₃ cathode, the full Na-ion cell delivers a remarkable energy density of 98 Wh kg⁻¹ (based on the mass of anode and cathode) and retains 80% of its capacity at 5C rate over 1000 cycles. The study opens new possibilities for enhancing the electrochemical performance of NASICON anodes via chemical and structural modulations.