Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.     

Metagenomic era for biocatalyst identification

Microbial enzymes have many known applications as biocatalysts. However, only a few of them are currently employed for biocatalysis even though an annotated collection of more than 190 billion bases is available in metagenome sequence databases from uncultured and highly diverse microbial populations. This review aims at providing conceptual and technical bases for the translation of metagenome data into both experimental and computational frameworks that facilitates a comprehensive analysis of the biocatalysts diversity space. We will also briefly present the status of the current capabilities that assess and predict catalytic potential of environmental sites and track its diversity and evolution in large-scale biocatalysis process resulting from studies applying metagenomics in association with gene fingerprinting, catabolic arrays and complementary '-omics'.     

Diversity of Glycosyl Hydrolases from Cellulose-Depleting Communities Enriched from Casts of Two Earthworm Species

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).Microorganisms producing diverse glycosyl hydrolases (GHs) are widespread and typically thrive in environments where plant materials tend to accumulate and deteriorate (42, 73). The habitats of microorganisms with great GH diversity are the ruminant animal rumen, mouse bowel, and rabbit cecum (10, 24, 26, 28, 49, 74). Microorganisms associated with soil invertebrates in general and with soil earthworms in particular carry out metabolic processes that contribute to element cycling and are essential in sustaining processes which their hosts are unable to perform (20, 52, 72, 76). Although some species of earthworms produce cellulases (15, 55), they generally rely on microbes inhabiting their gastrointestinal (GI) tracts to perform cellulose utilization processes (31, 47, 77). Casts are of special interest in this respect. Considering that the overall numbers of cellulolytic microbes in earthworm casts are greater than those in soil (57), earthworm casts seem to play an important role in the decomposition of plant litter, serving as an inoculum for cellulosic substrates (9). It is important to note that microorganisms from preingested substratum (soil or plant litter) are predominant in the gut lumen (20); however, microbial populations in earthworm casts differ from those in soil in terms of diversity and the relative abundance of different taxa (29, 57, 63). It is anticipated that the enzymatic repertoire of such microbial communities must be especially broad toward diverse sugar-based polymeric, oligomeric, and monomeric substrates; however, among approximately 115 families of GHs with thousands of members known to date (12), none of the GHs have been derived from microorganisms of earthworm-associated microbial communities.The aim of the present work was therefore to examine the diversity of GHs in metagenome libraries derived from fresh casts of Aporrectodea caliginosa and Lumbricus terrestris earthworms via functional screening. Other important tasks of this work were to characterize individual enzymes and to gain insight into their structural-functional features. Finally, we performed sequence analysis of large contiguous DNA fragments of fosmids harboring the genes for GHs to associate them with the organism(s) that may produce them, which was complemented by conventional small-subunit (SSU) rRNA clone library sequencing analysis.     

Genomic signatures of fifth autotrophic carbon assimilation pathway in bathypelagic Crenarchaeota

Marine Crenarchaeota, ubiquitous and abundant organisms in the oceans worldwide, remain metabolically uncharacterized, largely due to their low cultivability. Identification of candidate genes for bicarbonate fixation pathway in the Cenarchaeum symbiosum A was an initial step in understanding the physiology and ecology of marine Crenarchaeota. Recent cultivation and genome sequencing of obligate chemoautotrophic Nitrosopumilus maritimus SCM1 were a major breakthrough towards understanding of their functioning and provide a valuable model for experimental validation of genomic data. Here we present the identification of multiple key components of 3-hydroxipropionate/4-hydroxybutyrate cycle, the fifth pathway in carbon fixation, found in data sets of environmental sequences representing uncultivated superficial and bathypelagic Crenarchaeota from Sargasso sea (GOS data set) and KM3 (Mediterranean Sea) and ALOHA (Atlantic ocean) stations. These organisms are likely to use acetyl-CoA/propionyl-CoA carboxylase(s) as CO₂-fixing enzyme(s) to form succinyl-CoA, from which one molecule of acetyl-CoA is regenerated via 4-hydroxybutyrate cleavage and another acetyl-CoA to be the pathway product. The genetic distinctiveness and matching sympatric abundance imply that marine crenarchaeal genotypes from the three different geographic sites share similar ecophysiological properties, and therefore may represent fundamental units of marine ecosystem functioning. To couple results of sequence comparison with the dark ocean primary production, dissolved inorganic carbon fixation rates were measured at KM3 Station (3000 m depth, Eastern Mediterranean Sea), i.e. at the same site and depth used for metagenomic library construction.     

Inter‐conversion of catalytic abilities in a bifunctional carboxyl/feruloyl‐esterase from earthworm gut metagenome

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k_cat/K_m)]_CE/[(k_cat/K_m)]_FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys²⁸¹, Asp²⁸², Asn³¹⁶ and Lys³¹⁷) situated close to the catalytic core (Ser¹⁴³/Asp²⁷³/His³⁰⁵) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k_cat/K_m values) and enzymes with inverted specificity ((k_cat/K_m)_CE/(k_cat/K_m)_FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol⁻¹), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol⁻¹) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.     

Biodistribution and long-term fate of silver nanoparticles functionalized with bovine serum albumin in rats

Silver nanocrystals (Ag NCs) hold promising antibiotic and antiviral properties in biological systems. The biodistribution of silver nanostructures injected into animals in vivo is currently unknown, remaining as a fundamental issue for potential therapeutic applications. Here, we injected Ag NCs capped with bovine serum albumin (BSA) in live rats to elucidate their fate in several organs including liver, heart and brain. Very significant accumulations of nanoparticles were confirmed by inductively coupled plasma mass spectroscopy (ICPMS) and transmission electron microscopy (TEM) techniques on the liver and heart. In contrast, the brain tissue did not reveal evidence of particles content. Our results suggest that Ag+ permeated across the blood-brain barrier (BBB), and followed swift clearance from the organ.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Effect of the Immobilization Method of Lipase from Thermomyces lanuginosus on Sucrose Acylation

Lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa ) was immobilized using granulation by incubating low-particle-size silica with the lipase. Granules with a particle diameter in the range 0.3-1 &#117 mm were obtained. The immobilized lipase was tested in the acylation of sucrose with vinyl laurate in mixtures of tert -amyl alcohol: dimethyl sulfoxide. Results were compared with immobilization of enzyme by adsorption on polypropylene (Accurel EP100), deposition on Celite by precipitation, and covalent attachment to Eupergit C. Granulated lipase converted >95% of sucrose into 6- O -lauroylsucrose in 6 &#117 h. Accurel-lipase was also very active, converting 70% of sucrose into monoester in 2 &#117 h. The residual activity of granules after five reaction cycles under the best reaction conditions was 72%; this value was considerably higher than the one observed for the same lipase adsorbed on Accurel (15% residual activity after five cycles).     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.