Enhanced enzymatic production of cholesteryl 6ʹ-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori

Background During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6ʹ-O-acyl-α-d-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. Methods We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. Results The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. Conclusions Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

Hibernating brown bears are protected against atherogenic dyslipidemia

To investigate mechanisms by which hibernators avoid atherogenic hyperlipidemia during hibernation, we assessed lipoprotein and cholesterol metabolisms of free-ranging Scandinavian brown bears (Ursus arctos). In winter- and summer-captured bears, we measured lipoprotein sizes and sub-classes, triglyceride-related plasma-enzyme activities, and muscle lipid composition along with plasma-levels of antioxidant capacities and inflammatory markers. Although hibernating bears increased nearly all lipid levels, a 36%-higher cholesteryl-ester transfer-protein activity allowed to stabilize lipid composition of high-density lipoproteins (HDL). Levels of inflammatory metabolites, i.e., 7-ketocholesterol and 11ß-prostaglandin F2α, declined in winter and correlated inversely with cardioprotective HDL2b-proportions and HDL-sizes that increased during hibernation. Lower muscle-cholesterol concentrations and lecithin-cholesterol acyltransferase activity in winter suggest that hibernating bears tightly controlled peripheral-cholesterol synthesis and/or release. Finally, greater plasma-antioxidant capacities prevented excessive lipid-specific oxidative damages in plasma and muscles of hibernating bears. Hence, the brown bear manages large lipid fluxes during hibernation, without developing adverse atherogenic effects that occur in humans and non-hibernators.

Rational Design for Enhanced Acyltransferase Activity in Water Catalyzed by the Pyrobaculum calidifontis VA1 Esterase

Biocatalytic transesterification is commonly carried out employing lipases in anhydrous organic solvents since hydrolases usually prefer hydrolysis over acyl transfer in bulk water. However, some promiscuous acyltransferases can catalyze acylation in an aqueous solution. In this study, a rational design was performed to enhance the acyltransferase selectivity and substrate scope of the Pyrobaculum calidifontis VA1 esterase (PestE). PestE wild type and variants were applied for the acylation of monoterpene alcohols. The mutant PestE_I208A is selective for (–)-menthyl acetate (E-Value = 55). Highly active acyltransferases were designed, allowing for complete conversion of (–)-citronellol to citronellyl acetate. Additionally, carvacrol was acetylated but with lower conversions. To the best of our knowledge, this is the first example of the biocatalytic acylation of a phenolic alcohol in bulk water. In addition, a high citronellol conversion of 92% was achieved with the more environmentally friendly and inexpensive acyl donor ethyl acetate using PestE_N288F as a catalyst. PestE_N288F exhibits good acyl transfer activity in an aqueous medium and low hydrolysis activity at the same time. Thus, our study demonstrates an alternative synthetic strategy for acylation of compounds without organic solvents.

Comparative Investigation of Fifteen Xenobiotic Metabolizing N -Acetyltransferase (NAT) Homologs from Bacteria

Arylamines constitute a large group of industrial chemicals detoxified by certain bacteria through conjugation reactions catalyzed by N -acetyltransferase (NAT) enzymes. NAT homologs, mostly from pathogenic bacteria, have been the subject of individual studies that do not facilitate direct comparisons. By implementing a practicable pipeline, we present comparative investigation of fifteen NAT homologs from ten bacteria, mainly bacilli, streptomycetes, and one alphaproteobacterium. The new homologs were characterized for their sequence, phylogeny, predicted structural features, substrate specificity, thermal stability, and interaction with components of the enzymatic reaction. Bacillus NATs demonstrated the characteristics of xenobiotic metabolizing N -acetyltransferases, with the majority of homologs generating high activities. Non-pathogenic bacilli are thus proposed as suitable mediators of arylamine bioremediation. Of the Streptomyces homologs, the NAT2 isoenzyme of S. venezuelae efficiently transformed highly toxic arylamines, while the remaining homologs were inactive or generated low activities suggesting that xenobiotic metabolism may not be their primary role. The functional divergence of Streptomyces NATs was consistent with their observed sequence, phylogenetic, and structural variability. These and previous findings support classification of microbial NATs into three groups. The first includes xenobiotic metabolizing enzymes with dual acetyl-/propionyl-CoA selectivity. Homologs of the second group are more rarely encountered, acting as malonyltransferases mediating specialized ecological interactions. Homologs of the third group effectively lack acyltransferase activity and their study may represent an interesting research area. Comparative NAT enzyme screens from a broad microbial spectrum may guide rational selection of homologs likely to share similar biological functions, allowing their combined investigation and use in biotechnological applications. IMPORTANCE Arylamines are encountered as industrial chemicals or byproducts of agrochemicals that may constitute highly toxic contaminants of soils and groundwaters. Although such chemicals may be recalcitrant to biotransformation, they can be enzymatically converted into less toxic forms by some bacteria. Therefore, exploitation of the arylamine detoxification capabilities of microorganisms is investigated as an effective approach for bioremediation. Among microbial biotransformations of arylamines, enzymatic conjugation reactions have been reported, including NAT-mediated N- acetylation. Comparative investigations of NAT enzymes across a range of microorganisms can be laborious and expensive, so here we present a streamlined methodology for implementing such work. We compare fifteen NAT homologs from non-pathogenic, free-living bacteria of potential biotechnological utility, mainly Terrabacteria known for their rich secondary and xenobiotic metabolism. The analysis allowed insights into the evolutionary and functional divergence of bacterial NAT homologs, combined with assessment of their fundamental structural and enzymatic differences and similarities.

S-acylation of P2K1 mediates extracellular ATP-induced immune signaling in Arabidopsis

S-acylation is a reversible protein post-translational modification mediated by protein S-acyltransferases (PATs). How S-acylation regulates plant innate immunity is our main concern. Here, we show that the plant immune receptor P2K1 (DORN1, LecRK-I.9; extracellular ATP receptor) directly interacts with and phosphorylates Arabidopsis PAT5 and PAT9 to stimulate their S-acyltransferase activity. This leads, in a time-dependent manner, to greater S-acylation of P2K1, which dampens the immune response. pat5 and pat9 mutants have an elevated extracellular ATP-induced immune response, limited bacterial invasion, increased phosphorylation and decreased degradation of P2K1 during immune signaling. Mutation of S-acylated cysteine residues in P2K1 results in a similar phenotype. Our study reveals that S-acylation effects the temporal dynamics of P2K1 receptor activity, through autophosphorylation and protein degradation, suggesting an important role for this modification in regulating the ability of plants in respond to external stimuli.

Identification and Characterization of a Novel Carboxylesterase EstQ7 from a Soil Metagenomic Library

A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174–Asp306–His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910 S-1, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Homology modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking reveled the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property, and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

Versatility in acyltransferase activity completes chicoric acid biosynthesis in purple coneflower

Purple coneflower (Echinacea purpurea (L.) Moench) is a popular native North American herbal plant. Its major bioactive compound, chicoric acid, is reported to have various potential physiological functions, but little is known about its biosynthesis. Here, taking an activity-guided approach, we identify two cytosolic BAHD acyltransferases that form two intermediates, caftaric acid and chlorogenic acid. Surprisingly, a unique serine carboxypeptidase-like acyltransferase uses chlorogenic acid as its acyl donor and caftaric acid as its acyl acceptor to produce chicoric acid in vacuoles, which has evolved its acyl donor specificity from the better-known 1-O-β-D-glucose esters typical for this specific type of acyltransferase to chlorogenic acid. This unusual pathway seems unique to Echinacea species suggesting convergent evolution of chicoric acid biosynthesis. Using these identified acyltransferases, we have reconstituted chicoric acid biosynthesis in tobacco. Our results emphasize the flexibility of acyltransferases and their roles in the evolution of specialized metabolism in plants.

Impact of Phosphate Availability on Membrane Lipid Content of the Model Strains, Streptomyces lividans and Streptomyces coelicolor

In this issue we demonstrated that the phospholipid content of Streptomyces lividans varies greatly with Pi availability being was much lower in Pi limitation than in Pi proficiency whereas that of Streptomyces coelicolor varied little with Pi availability. In contrast the content in phosphate free ornithine lipids was enhanced in both strains in condition of phosphate limitation. Ornithine lipids biosynthesis starts with the N-acylation of ornithine to form lyso-ornithine that is then O-acylated to yield ornithine lipid. The operon sco1222-23 was proposed to be involved in the conversion of specific amino acids into ornithine in condition of phosphate limitation whereas the sco0921-20 operon encoding N- and O-acyltransferase, respectively, was shown to be involved in the biosynthesis of these lipids. The expression of these two operons was shown to be under the positive control of the two components system PhoR/PhoP and thus induced in phosphate limitation. The expression of phoR/phoP being weak in S. coelicolor, the poor expression of these operons resulted into a fivefold lower ornithine lipids content in this strain compared to S. lividans. In the deletion mutant of the sco0921-20 operon of S. lividans, lyso-ornithine and ornithine lipids were barely detectable and TAG content was enhanced. The complementation of this mutant by the sco0921-20 operon or by sco0920 alone restored ornithine lipids and TAG content to wild type level and was correlated with a twofold increase in the cardiolipin content. This suggested that SCO0920 bears, besides its broad O-acyltransferase activity, an N-acyltransferase activity and this was confirmed by the detection of lyso-ornithine in this strain. In contrast, the complementation of the mutant by sco0921 alone had no impact on ornithine lipids, TAG nor cardiolipin content but was correlated with a high lyso-ornithine content. This confirmed that SCO0921 is a strict N-acyltransferase. However, interestingly, the over-expression of the sco0921-20 operon or of sco0921 alone in S. coelicolor, led to an almost total disappearance of phosphatidylinositol that was correlated with an enhanced DAG and TAG content. This suggested that SCO0921 also acts as a phospholipase C, degrading phosphatidylinositol to indirectly supply of phosphate in condition of phosphate limitation.