Characterization of a Novel Esterase Est33 From an Antarctic Bacterium: A Representative of a New Esterase Family.

Studies of microorganisms from extreme environments can sometimes reveal novel proteins with unique properties. Here, we identified a novel esterase gene (Est33) from an Antarctic bacterium. The protein was expressed and purified for biochemical characterizations. Site-mutation variants including S94A, D205A, and H233A were constructed to explore the structure-function relationship of the catalytic triad of Est33, and we found mutating Ser94, Asp205, and His233 residues lead to a complete loss of enzyme activity. In addition, the catalytic Ser94 located in a conserved pentapeptide motif GVSWG. Phylogenetic analysis showed that Est33 and its closely related homologs belonged to an independent group apart from other known family members, indicating that Est33 represented a new family of esterase. The Est33 enzyme was found to be a cold-active esterase retaining 25%-100% activity from 10°C to 30°C and to have optimal catalytic activity toward p-nitrophenol acetate (30°C and pH7.5). The serine modifying reagent phenylmethylsulfonyl fluoride inhibited the activity of Est33 by 77.34%, while thiol reagents such as dithiol threitol (DTT) activated the enzyme by 3-fold. Metal chelating reagents EDTA had no effects, indicating that Est33 is not a metalloenzyme. Collectively, these results indicate that Est33 constitutes the first member of a novel esterase family XXI that has been identified.

Identification and Biochemical Characterization of a Novel Hormone-Sensitive Lipase Family Esterase Est19 from the Antarctic Bacterium Pseudomonas sp. E2-15.

Esterases represent an important class of enzymes with a wide variety of industrial applications. A novel hormone-sensitive lipase (HSL) family esterase, Est19, from the Antarctic bacterium Pseudomonas sp. E2-15 is identified, cloned, and expressed. The enzyme possesses a GESAG motif containing an active serine (S) located within a highly conserved catalytic triad of Ser155, Asp253, and His282 residues. The catalytic efficiency (kcat/Km) of Est19 for the pNPC6 substrate is 148.68 s-1mM-1 at 40 °C. Replacing Glu154 juxtaposed to the critical catalytic serine with Asp (E154→D substitution) reduced the activity and catalytic efficiency of the enzyme two-fold, with little change in the substrate affinity. The wild-type enzyme retained near complete activity over a temperature range of 10-60 °C, while ~50% of its activity was retained at 0 °C. A phylogenetic analysis suggested that Est19 and its homologs may represent a new subfamily of HSL. The thermal stability and stereo-specificity suggest that the Est19 esterase may be useful for cold and chiral catalyses.

The benzoate plant metabolite ethyl gallate prevents cellular- and vascular-lipid accumulation in experimental models of atherosclerosis.

Ethyl gallate (EG) is a well-known constituent of medicinal plants, but its effects on atherosclerosis development are not clear. In the present study, the anti-atherosclerosis effects of EG and the underlying mechanisms were explored using macrophage cultures, zebrafish and apolipoprotein (apo) E deficient mice. Treatment of macrophages with EG (20 µM) enhanced cellular cholesterol efflux to HDL, and reduced net lipid accumulation in response to oxidized LDL. Secretion of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) from activated macrophages was also blunted by EG. Fluorescence imaging techniques revealed EG feeding of zebrafish reduced vascular lipid accumulation and inflammatory responses in vivo. Similar results were obtained in apoE-/- mice 6.5 months of age, where plaque lesions and monocyte infiltration into the artery wall were reduced by 70% and 42%, respectively, after just 6 weeks of injections with EG (20 mg/kg). HDL-cholesterol increased 2-fold, serum cholesterol efflux capacity increased by ∼30%, and the levels of MCP-1 and IL-6 were reduced with EG treatment of mice. These results suggest EG impedes early atherosclerosis development by reducing the lipid and macrophage-content of plaque. Underlying mechanisms appeared to involve HDL cholesterol efflux mechanisms and suppression of pro-inflammatory cytokine secretion.

Diversity of protease-producing bacteria in the soils of the South Shetland Islands, Antarctica.

Proteases secreted from bacteria into soil play a key role in the degradation of organic nitrogen, which is the first and, usually, the rate-limiting step of nitrogen cycling. However, the diversity of protease-producing bacteria and their excreted proteases in Antarctic soil have not yet been fully explored. Here we studied 20 soil samples from the South Shetland Islands, Antarctica and isolated 253 strains with protease activity. These protease-producing bacteria belonged to the phyla Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes, and Deinococcus-Thermus. Thhe predominant genera were Arthrobacter (14.9%), Chryseobacterium (14.5%), Flavobacterium (14.5%), and Pseudomonas (14.5%). Most of these bacteria secreted serine proteases and metalloproteases. There was quite a large distribution in activity as quantified by protease and inhibition assays. Only a few strains secreted aspartic and/or cysteine proteases. Together these data provided novel insight into the diversity and mechanism of organic nitrogen degradation in Antarctic soils by various proteases, which may have potential in new biotechnological applications.