Structure and Mechanism of the Bifunctional CinA Enzyme from Thermus thermophilus

CinA is a widely distributed protein in Gram-positive and Gram-negative bacteria. It is associated with natural competence and is proposed to have a function as an enzyme participating in the pyridine nucleotide cycle, which recycles products formed by non-redox uses of NAD. Here we report the determination of the crystal structure of CinA from Thermus thermophilus, in complex with several ligands. CinA was shown to have both nicotinamide mononucleotide deamidase and ADP-ribose pyrophosphatase activities. The crystal structure shows an unusual asymmetric dimer, with three domains for each chain; the C-terminal domain harbors the nicotinamide mononucleotide deamidase activity, and the structure of a complex with the product nicotinate mononucleotide suggests a mechanism for deamidation. The N-terminal domain belongs to the COG1058 family and is associated with the ADP-ribose pyrophosphatase activity. The asymmetry in the CinA dimer arises from two alternative orientations of the COG1058 domains, only one of which forms a contact with the KH-type domain from the other chain, effectively closing the active site into, we propose, a catalytically competent state. Structures of complexes with Mg²⁺/ADP-ribose, Mg²⁺/ATP, and Mg²⁺/AMP suggest a mechanism for the ADP-ribose pyrophosphatase reaction that involves a rotation of the COG1058 domain dimer as part of the reaction cycle, so that each active site oscillates between open and closed forms, thus promoting catalysis.     

Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

In cynobacteria and higher plants, salinity is known to inhibit the activity of several enzymes involved in photosynthesis and hence decreases the overall photosynthetic rate. This gave us an impetus to search for a protease, which may be involved in the turnover of non-functional enzymes produced under salinity stress. Taking the possible changes in pH gradient of the chloroplast under consideration, we have tried to identify a protease, which is induced under salinity and characterized it as an alkaline protease using spinach (Spinacia oleracea) leaves as a model system. The HIC-HPLC purified homogeneous alkaline serine protease from the isolated spinach chloroplasts had two subunits of molecular weight 63 and 32 kDa. The enzyme was maximally active at pH 8.5 and 50°C. The enzyme showed the property to hydrolyze the synthetic substrate like azocaesin and had sufficient proteolytic activity in gelatin bound native PAGE. The enzyme activity was also dependent upon the presence of divalent cations and reduced environment. The active site residues were identified and the homogeneous alkaline serine protease had cysteine, lysine and tryptophan residues at its active site.     

Outer membrane translocons: structural insights into channel formation

Gram-negative bacteria need to maintain the integrity of their outer membrane while also regulating the secretion of toxins and other macromolecules. A variety of dedicated outer membrane proteins (OMPs) facilitate this process. Recent structural work has shown that some of these proteins adopt classical β-barrel transmembrane structures and rely on structural changes within the barrel lumen to allow passage of substrate proteins. Other secretion systems have OMP components which use transmembrane α-helices and appear to function in a different way. Here we review a selection of recent structural studies which have major ramifications for our understanding of the passage of macromolecules across the outer membrane.     

Characterization of a unique glycosylated anchor endopeptidase that cleaves the LPXTG sequence motif of cell surface proteins of Gram-positive bacteria

The precursors of most surface proteins on Gram-positive bacteria have a C-terminal hydrophobic domain and charged tail, preceded by a conserved LPXTG motif that signals the anchoring process. This motif is the substrate for an enzyme, termed sortase, which has transpeptidation activity resulting in the cleavage of the LPXTG sequence and ultimate attachment of the protein to the peptidoglycan. While screening a group A streptococcal membrane extract for cleavage activity of the LPXTG motif, we identified an enzyme (which we term "LPXTGase") that differs significantly from sortase but also cleaves this motif. The enzyme is heavily glycosylated, which is required for its activity. Amino acid composition and sequence analysis revealed that LPXTGase differs from other enzymes, in that the molecule, which is about 14 kDa in size, has no aromatic amino acids, is rich in alanine, and is 30% composed of uncommon amino acids, suggesting a nonribosomal construction. A similar enzyme found in the membrane extract of Staphylococcus aureus, indicates that this unusual molecule may be common among Gram-positive bacteria. Whereas peptide antibiotics have been reported from bacillus species that also contain unusual amino acids and are synthesized non-ribosomally on amino acid-activating polyenzyme templates, this would be the first reported enzyme that may be similarly synthesized.     

Mating compatibility among helicoverpa armigera (Lepidoptera: Noctuidae) occurring on selected host plants and Bt cotton survivors

Helicoverpa armigera (Hiibner) (Lepidoptera: Noctuidae) is a well-known polyphagous insect pest. Mating compatibility among the insects occurring on different host plants is essential for free gene flow among populations. We tested the extent of crossability and fecundity of the insects that survived on Bacillus thuringiensis (Bt) cotton with those occurring on pigeon pea, Cajanus cajun (L.) Millsp., non-Bt cotton, Gossypium hirsutum L.; sunflower, Helianthus annuus; sorghum, Sorghum bicolor L. Moench.; okra, Abelmoschus moschatus Medikus; chickpea, Cicer arietinum L.; marigold, Tagetes spp.; and tomato, Lycopersicum esculentum L., crops. The insects from different crops were freely crossable with those collected from Bt cotton and among themselves. The average fecundity across different crosses ranged from 314.1 to 426.3 in direct and from 305.8 to 421.7 eggs per female in reciprocal crosses. In any given cross, a minimum of 85.89% egg hatch was recorded. Furthermore, the F1 individuals of different cross combinations were found to cross freely with their parents (BC1) and among themselves with similar fecundity and egg hatch. High crossability among H. armigera occurring on different host plants suggests that crop mosaics that may exist in countries such as India could play an important role as natural, nonstructured refugia and prolong the durability of the genes deployed for controlling this insect.     

Generation and characterization of DSPP‐Cerulean/DMP1‐Cherry reporter mice

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3‐GFP, pOBCol3.6‐GFP, and DMP1‐GFP), similar to the endogenous proteins, are also expressed by bone‐forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP‐Cerulean/DMP1‐Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24‐258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1‐Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP‐Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.     

Hierarchically Multiscale Vertically Oriented NiFeCo Nanoflakes for Efficient Electrochemical Oxygen Evolution at High Current Densities

Crucial advancements in versatile catalyst systems capable of achieving high current densities under industrial conditions, bridging the gap between fundamental understanding and practical applications, are pivotal to propel the hydrogen economy forward. In this study, vertically oriented hierarchically multiscale nanoflakes of NiFeCo electrocatalysts are presented, developed by surface modification of a porous substrate with nano-structured nickel. The resulting electrodes achieve remarkably low overpotentials of 139 mV at 10 mAcm⁻² and 248 mV at 500 mAcm⁻². Further, scaled-up electrodes are implemented in a water-splitting electrolyser device exhibiting a stable voltage of 1.82 V to deliver a constant current density of 500 mA cm⁻² for over 17 days. Moreover, the role of the unique structures on electrochemical activity is systematically investigated by fractal analysis, involving computation of structure factors such as Minkowski connectivity, fractal dimension, and porosity using scanning electron microscope images. It is found that such structures offer higher surface area than typical layered double hydroxide structures due to morphological coherence that results in a superhydrophilic surface, while the base Ni layer boosts the charge transfer. This study demonstrates a Ni/NiFeCo(OH)_x heterostructure with highly porous morphology, a key to unlocking extremely efficient oxygen evolution reaction activity with exceptional stability. Moreover, fractal analysis is presented as a valuable tool to evaluate the electrochemical performance of catalysts for their structured morphology.     

MLT-10 Defines a Family of DUF644 and Proline-rich Repeat Proteins Involved in the Molting Cycle of Caenorhabditis elegans

The molting cycle of nematodes involves the periodic synthesis and removal of a collagen-rich exoskeleton, but the underlying molecular mechanisms are not well understood. Here, we describe the mlt-10 gene of Caenorhabditis elegans, which emerged from a genetic screen for molting-defective mutants sensitized by low cholesterol. MLT-10 defines a large family of nematode-specific proteins comprised of DUF644 and tandem P-X₂-L-(S/T)-P repeats. Conserved nuclear hormone receptors promote expression of the mlt-10 gene in the hypodermis whenever the exoskeleton is remade. Further, a MLT-10::mCherry fusion protein is released from the hypodermis to the surrounding matrices and fluids during molting. The fusion protein is also detected in strands near the surface of animals. Both loss-of-function and gain-of-function mutations of mlt-10 impede the removal of old cuticles. However, the substitution mutation mlt-10(mg364), which disrupts the proline-rich repeats, causes the most severe phenotype. Mutations of mlt-10 are also associated with abnormalities in the exoskeleton and improper development of the epidermis. Thus, mlt-10 encodes a secreted protein involved in three distinct but interconnected aspects of the molting cycle. We propose that the molting cycle of C. elegans involves the dynamic assembly and disassembly of MLT-10 and possibly the paralogs of MLT-10.