Two SusD-like proteins encoded within a polysaccharide utilisation locus of an uncultured ruminant bacteroidetes bind strongly to cellulose

We demonstrate that two characteristic Sus-like proteins encoded within a Polysaccharide Utilisation Locus (PUL) bind strongly to cellulosic substrates and interact with plant primary cell walls. This shows associations between uncultured Bacteroidetes-affiliated lineages and cellulose in the rumen, and thus presents new PUL-derived targets to pursue regarding plant biomass degradation.

High-resolution refinement of orthorhombic bovine pancreatic phospholipase A2

The X-ray structure of recombinant bovine pancreatic phospholipase A(2) (PLA2), which specifically catalyzes the cleavage of the sn-2 acylester bond of phospholipids, has been refined at 1.5 Angstrom resolution. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a = 47.12, b = 64.59 and c = 38.14 Angstrom similar to the native enzyme reported previously by Dijkstra et nl. [J. Mel. Biol. (1981), 147, 97-123]. The refinement converged to an R value of 18.4% (R-free = 22.8%) for 16 374 reflections between 10.0 and 1.5 Angstrom resolution. The surface-loop residues (60-70) art: ordered in the present orthorhombic recombinant enzyme, but disordered in the trigonal recombinant enzyme. The active-site residues, His48, Asp99, and the catalytic water superimpose well with the trigonal form. Besides the catalytic water which is hydrogen bonded to His48, it is often seen that there is a second water attached to the same N atom of His48 and simultaneously hydrogen bonded to the O atom of Asp49. It is thought that the second water facilitates the tautomerism of His48 for enzyme catalysis, The catalytic water is also hydrogen bonded to the equatorial water coordinated to the calcium ion, In addition to the equatorial water, there is also an axial calcium water and the additional structural water. These five common water molecules are hydrogen bonded to the additional 16 water molecules in the present orthorhombic structure which may further enhance the structural integrity of the active site. Besides the protein and one calcium ion, a total of 134 water molecules were located in the present high-resolution refinement.

Binding of active site directed ligands to phospholipase A2: Implications on the molecular constraints and catalytic mechanism

Molecular constraints for the localization of active site directed ligands (competitive inhibitors and substrates) in the active site of phospholipase A2 (PLA2) are characterized. Structure activity relationships with known inhibitors suggest that the head : group interactions dominate the selectivity as well as a substantial part of the affinity. The ab initio fitting of the amide ligands in the active site was carried out to characterize the head group interactions. Based on a systematic coordinate space search, formamide is docked with known experimental constraints such as coordination of the carbonyl group to Ca2+ and hydrogen bond between amide nitrogen and ND1 of His48. An optimal position for a bound water molecule is identified and its significance for the catalytic mechanism is postulated. Unlike the traditional ''pseudo-triad'' mechanism, the ''Ca-coordinatedoxyanion'' mechanism proposed here invokes activation of the catalytic water to form the oxyanion in the coordination sphere of calcium. As it attacks the carbonyl carbon of the ester, a near-tetrahedral intermediate is formed. As the second proton of the catalytic water is abstracted by the ester oxygen, its reorientation and simultaneous cleavage form hydrogen bond with ND1 of His48. In this mechanism of esterolysis, a catalytic role for the water co-ordinated to Ca2+ is recognised.

Phylogenetic relationships and classification of thiolases and thiolase-like proteins of Mycobacterium tuberculosis and Mycobacterium smegmatis

Thiolases are enzymes involved in lipid metabolism. Thiolases remove the acetyl-CoA moiety from 3-ketoacyl-CoAs in the degradative reaction. They can also catalyze the reverse Claisen condensation reaction, which is the first step of biosynthetic processes such as the biosynthesis of sterols and ketone bodies. In human, six distinct thiolases have been identified. Each of these thiolases is different from the other with respect to sequence, oligomeric state, substrate specificity and subcellular localization. Four sequence fingerprints, identifying catalytic loops of thiolases, have been described. In this study genome searches of two mycobacterial species (Mycobacterium tuberculosis and Mycobacterium smegmatis), were carried out, using the six human thiolase sequences as queries. Eight and thirteen different thiolase sequences were identified in M. tuberculosis and M. smegmatis, respectively. In addition, thiolase-like proteins (one encoded in the Mtb and two in the Msm genome) were found. The purpose of this study is to classify these mostly uncharacterized thiolases and thiolase-like proteins. Several other sequences obtained by searches of genome databases of bacteria, mammals and the parasitic protist family of the Trypanosomatidae were included in the analysis. Thiolase-like proteins were also found in the trypanosomatid genomes, but not in those of mammals. In order to study the phylogenetic relationships at a high confidence level, additional thiolase sequences were included such that a total of 130 thiolases and thiolase-like protein sequences were used for the multiple sequence alignment. The resulting phylogenetic tree identifies 12 classes of sequences, each possessing a characteristic set of sequence fingerprints for the catalytic loops. From this analysis it is now possible to assign the mycobacterial thiolases to corresponding homologues in other kingdoms of life. The results of this bioinformatics analysis also show interesting differences between the distributions of M. tuberculosis and M. smegmatis thiolases over the 12 different classes. (C) 2014 Elsevier Ltd. All rights reserved.

Ca2+-independent phospholipase A2-dependent gating of TRPM8 by lysophospholipids

TRPM8 represents an ion channel activated by cold temperatures and cooling agents, such as menthol, that underlies the cold-induced excitation of sensory neurons. Interestingly, the only human tissue outside the peripheral nervous system, in which the expression of TRPM8 transcripts has been detected at high levels, is the prostate, a tissue not exposed to any essential temperature variations. Here we show that the TRPM8 cloned from human prostate and heterologously expressed in HEK-293 cells is regulated by the Ca(2+)-independent phospholipase A(2) (iPLA(2)) signaling pathway with its end products, lysophospholipids (LPLs), acting as its endogenous ligands. LPLs induce prominent prolongation of TRPM8 channel openings that are hardly detectable with other stimuli (e.g. cold, menthol, and depolarization) and that account for more than 90% of the total channel open time. Down-regulation of iPLA(2) resulted in a strong inhibition of TRPM8-mediated functional responses and abolished channel activation. The action of LPLs on TRPM8 channels involved either changes in the local lipid bilayer tension or interaction with the critical determinant(s) in the transmembrane channel core. Based on this, we propose a novel concept of TRPM8 regulation with the involvement of iPLA(2) stimulation. This mechanism employs chemical rather than physical (temperature change) signaling and thus may be the main regulator of TRPM8 activation in organs not exposed to any essential temperature variations, as in the prostate gland.

Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica.

Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.

Cloning of cDNAs and molecular characterisation of C-type lectin-like proteins from snake venoms

C-type lectin-like proteins (CTLPs) isolated from snake venoms are the largest and most complex non-mammalian vertebrate C-type lectin-like domain family. In the present study, we simultaneously amplified four cDNAs encoding different types of CTLP subunits from the venoms of two different species of snakes by RT-PCR with a single sense primer and a nested universal primer - two CTLP subunit-encoding cDNAs were cloned from Deinagkistrodon acutus venom and two from Agkistrodon halys Pallas venom. All four cloned CTLP subunits exhibited typical motifs in their corresponding domain regions but with relatively-low sequence similarities to each other. Compared with previously-published CTLPs, the four cloned CTLPs subunits showed slight variations in the calcium-binding sites and the disulphide bonding patterns. To our knowledge, these data constitute the first example of co-expression of CTLP platelet glycoprotein Ib-binding subunits and coagulation factors in Agkistrodon halys Pallas venom.

Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells

We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications.