Bacterial Esterase Research Articles

Ester derivatives of Dictyostelium differentiation-inducing factors exhibit antibacterial activity, possibly via a prodrug-like function

ObjectiveDictyostelium differentiation-inducing factors 1 and 3 [DIF-1 (1) and DIF-3 (2), respectively], along with their derivatives, such as Ph-DIF-1 (3) and Bu-DIF-3 (4), demonstrate antibacterial activity in vitro against Gram-positive bacteria, including methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-sensitive Enterococcus faecalis (VSE), and vancomycin-resistant Enterococcus faecium [VRE (VanA)]. This study investigates the therapeutic potential of DIF compounds against these Gram-positive bacteria.ResultsIn vitro tests revealed that the antibacterial activity of 3 and 4 was lost in the presence of human serum albumin (HSA), suggesting that HSA might inhibit their effectiveness. Further evaluation of less hydrophobic derivatives, DIF-1-NH2 (5) and NH2-Bu-DIF-3 (6), showed no antibacterial activity, even in the absence of HSA. However, ester derivatives Ph-DIF-1(AHA) (7) and Bu-DIF-3(2Ac) (8) exhibited antibacterial activity against the target bacteria in vitro, although this activity was also lost in the presence of HSA. We hypothesize that these ester derivatives may function as prodrugs, with their antibacterial activity possibly restored by hydrolysis through bacterial esterases. The results suggest that suitable ester modifications could enhance the in vivo antibacterial potential of DIF compounds, particularly if they can bypass HSA binding and be activated by bacterial enzymes.

Effect of a chemically-modified-curcumin on dental resin biodegradation.

Previous studies have shown Streptococcus mutans (S. mutans) esterase is a key mediator of dental composite biodegradation, which can contribute to recurrent caries. This study is to investigate the inhibitory effects of a novel Chemically-Modified-Curcumin (CMC 2.24) on esterase activities and related dental material biodegradation. Dental adhesive materials and composite resins were incubated in S. mutans suspension with CMC 2.24 and other compounds, including doxycycline, Chemically-Modified-Tetracycline (CMT-3), and curcumin for 4 weeks. The pre- and post-incubation surface roughness were evaluated by either laser diffraction pattern and/or a 3D laser scanning microscope. Esterase enzyme inhibition assays were performed with the same test groups and activities were determined spectrophotometrically. Among all experimental groups, CMC 2.24 significantly reduced surface roughness of dental composite (p < 0.01) and adhesive (p < 0.01) materials compared to bacteria-only group. Additionally, CMC 2.24 reduced porcine esterase activity by 46.5%, while other compounds showed minimal inhibition. In the S. mutans esterase assay, CMC 2.24 showed inhibition of 70.0%, while other compounds showed inhibition ranging from 19% to 36%. Our study demonstrated that CMC 2.24 inhibited biodegradation of dental composite material more effectively than its mother compound, curcumin. Moreover, the mechanism of this biodegradation was likely mediated through bacterial esterase activity. Doxycycline achieved similar inhibition by completely eradicating S. mutans with its antibiotic action; hence, it is not recommended for long-term use.

Mechanistic and structural insights into EstS1 esterase: A potent broad-spectrum phthalate diester degrading enzyme.

Phthalate diesters are important pollutants and act as endocrine disruptors. While certain bacterial esterases have been identified for phthalate diesters degradation to monoesters, their structural and mechanistic characteristics remain largely unexplored. Here, we highlight the potential of the thermostable and pH-tolerant EstS1 esterase from Sulfobacillus acidophilus DSM10332 to degrade high molecular weight bis(2-ethylhexyl) phthalate (DEHP) by combining biophysical and biochemical approaches along with high-resolution EstS1 crystal structures of the apo form and with bound substrates, products, and their analogs to elucidate its mechanism. The catalytic tunnel mediates entry and exit of the substrate and product, respectively. The centralized Ser-His-Asp triad performs catalysis by a bi-bi ping-pong mechanism, forming a tetrahedral intermediate. Mutagenesis analysis showed that the Met207Ala mutation abolished DEHP binding at the active site, confirming its essential role in supporting catalysis. These findings underscore EstS1 as a promising tool for advancing technologies aimed at phthalate diesters biodegradation.

In-silico characterization of a hypothetical protein of Sulfobacillus sp. hq2 for degradation of phthalate diesters

Phthalate plasticizers are hazardous compounds capable of causing endocrine disruption, cancers, and developmental disorders. Phthalate diesters are commonly used plasticizers in plastic products (PVC pipes) that leach out into the environment due to changes in temperature, pressure, and pH, posing harmful effects on different life forms. Bioremediation of phthalate diesters utilizing bacterial esterase has been recognized as an efficient approach but few effective esterases capable of degrading a wide range of phthalate diesters have been identified. Further, the thermostability of these esterases is a highly desirable property for their applications in diverse in-situ conditions. In this present in-silico study a hypothetical protein (POB10642.1) as a high-potential esterase from a thermostable strain of Sulfobacillus sp. hq2 has been characterized. Analysis revealed a significant sequence identity of 42.67 % and structural similarity (RMSD 0.557) with known phthalate diester degrading EstS1 esterase and a high Tm range of 55–66 °C. Structural analysis revealed the presence of two cavities on the surface mediating toward the catalytic site forming a catalytic tunnel. The enzyme POB10642.1 has significant molecular docking binding energies in the range of −5.4 to −7.5 kcal/mol with several phthalate diesters, including Diethyl phthalate, Dipropyl phthalate, Dibutyl phthalate, Dipentyl phthalate, Dihexyl phthalate, Benzyl butyl phthalate, Dicyclohexyl phthalate, and Bis(2-ethylhexyl) phthalate. High stability of binding during 100 ns molecular dynamics simulations revealed efficient and stable binding of the enzyme with a wide range of phthalate diesters at its active site, demonstrating the ability of the identified esterase to interact with and degrade diverse phthalate diesters. Therefore, POB10642.1 esterase can be an efficient candidate to be utilized in the development of enzyme-based bioremediation technologies to reduce the toxic levels of phthalate diesters.

Abridgement of Microbial Esterases and Their Eminent Industrial Endeavors.

Esterases are hydrolases that contribute to the hydrolysis of ester bonds into both water-soluble acyl esters and emulsified glycerol-esters containing short-chain acyl groups. They have garnered significant attention from biotechnologists and organic chemists due to their immense commercial value. Esterases, with their diverse and significant properties, have become highly sought after for various industrial applications. Synthesized ubiquitously by a wide range of living organisms, including animals, plants, and microorganisms, these enzymes have found microbial esterases to be the preferred choice in industrial settings. The cost-effective production of microbial esterases ensures higher yields, unaffected by seasonal variations. Their applications span diverse sectors, such as food manufacturing, leather tanneries, paper and pulp production, textiles, detergents, cosmetics, pharmaceuticals, biodiesel synthesis, bioremediation, and waste treatment. As the global trend shifts toward eco-friendly and sustainable practices, industrial processes are evolving with reduced waste generation, lower energy consumption, and the utilization of biocatalysts derived from renewable and unconventional raw materials. This review explores the background, structural characteristics, thermostability, and multifaceted roles of bacterial esterases in crucial industries, aiming to optimize and analyze their properties for continued successful utilization in diverse industrial processes. Additionally, recent advancements in esterase research are overviewed, showcasing novel techniques, innovations, and promising areas for further exploration.

Flow cytometric assessments of metabolic activity in bacterial assemblages provide insight into ecosystem condition along the Buffalo National River, Arkansas

The Buffalo National River (BNR), on karst terrain in Arkansas, is considered an extraordinary water resource. Water collected in Spring 2017 along BNR was metagenomically analyzed using 16S rDNA, and for 17 months (5/2017–11/2018), bacterial responses were measured in relation to nutrients sampled along a stretch of BNR near a concentrated animal feed operation (CAFO) on Big Creek. Because cell count and esterase activity can increase proportionally with organic enrichment, they were hypothesized to be elevated near the CAFO. Counts (colony forming units; CFUs) were different among sites for 73 % of the months; Big Creek generated highest CFUs 27 % of the time, with the closest downstream site at 13.3 %. Esterase activity was different among sites 94 % of the time, with Big Creek exhibiting lowest activity 71 % of the time. Over the months, activity was similar across sites at ~70 % active, except at Big Creek (56 %). The α-diversity of BNR microbial consortia near a wastewater treatment plant (WWTP) and the CAFO was related to distance from the WWTP and CAFO. The inverse relationship between high CFUs and low esterase activity at Big Creek (r = −0.71) actuated in vitro exposures of bacteria to organic wastewater contaminants (OWC) previously identified in the watershed. Exponential-phase Escherichia coli (stock strain), Streptococcus suis (avirulent, from swine), and S. dysgalactiae (virulent, from silver carp, Hypophthalmichthys molitrix) were incubated with atrazine, pharmaceuticals (17 α-ethynylestradiol and trenbolone), and antimicrobials (tylosin and butylparaben). Bacteria were differentially responsive. Activity varied with exposure time and OWC type, but not concentration; atrazine decreased it most. Taken together - the metagenomic taxonomic similarities along BNR, slightly higher bacterial growth and lower bacterial esterase at the CAFO, and the lab exposures of bacterial strains showing that OWC altered metabolism - the results indicated that bioactive OWC entering the watershed can strongly influence microbial processes in the aquatic ecosystem.

Biochemical characterization, substrate and stereoselectivity of an outer surface putative α/β hydrolase from the pathogenic Leptospira

The genome of pathogenic leptospira encodes a plethora of outer surface and secretory proteins. The outer surface or secreted α/β hydrolases in a few pathogenic organisms are crucial virulent factors. They hydrolyze host immune factors and pathogen's immune-activating ligands, which help pathogens to evade the host's innate immunity. In this study, we report biochemical characterizations, substrate and stereoselectivity of one of the leptospiral outer surface putative α/β hydrolases, IQB77_09235 (LABH). Purified LABH displayed better kinetic parameters towards small water-soluble esters such as p-nitrophenyl acetate and p-nitrophenyl butyrate. The LABH exhibited moderate thermostability and displayed a pH optimum of 8.5. Remarkably, a phylogenetic study suggested that LABH does not cluster with other characterized bacterial esterases or lipases. Protein structural modeling revealed that some structural features are closely associated with Staphylococcus hycus lipase (SAH), a triacylglycerol hydrolase. The hydrolytic activity of the protein was found to be inhibited by a lipase inhibitor, orlistat. Biocatalytic application of the protein in the kinetic resolution of racemic 1-phenylethyl acetate reveals excellent enantioselectivity (E > 500) in the production of (R)-1-phenylethanol, a valuable chiral synthon in several industries. To our knowledge, this is the first detailed characterization of outer surface α/β hydrolases from leptospiral spp.

Functional and Transcriptome Analysis of Streptococcus pyogenes Virulence on Loss of Its Secreted Esterase.

Esterases are broadly expressed in bacteria, but much remains unknown about their pathogenic effect. In previous studies, we focused on an esterase secreted by Streptococcus pyogenes (group A Streptococcus, GAS). Streptococcal secreted esterase (Sse) can hydrolyze the sn−2 ester bonds of platelet−activating factor (PAF), converting it to an inactive form that inhibits neutrophil chemotaxis to the infection sites. However, as a virulent protein, Sse probably participates in GAS pathogenesis far beyond chemotaxis inhibition. In this study, we generated the sse gene knockout strain (Δsse) from the parent strain MGAS5005 (hypervirulent M1T1 serotype) and compared the difference in phenotypes. Absence of Sse was related to weakened skin invasion in a murine infection model, and significantly reduced GAS epithelial adherence, invasion, and intracellular survival. Reduced virulence of the Δsse mutant strain was explored through transcriptome analysis, revealing a striking reduction in the abundance of invasive virulence factors including M protein, SIC, ScpA, and SclA. Besides the influence on the virulence, Sse also affected carbohydrate, amino acid, pyrimidine, and purine metabolism pathways. By elucidating Sse−mediated pathogenic process, the study will contribute to the development of new therapeutic agents that target bacterial esterases to control clinical GAS infections.

Influence of Human and Bacterial Enzymes on Resin Restorations: A Review

Esthetic satisfaction has been a prime concern for patients. This has led to a surge in the development of esthetic restorations and dental composites in the field of restorative dentistry over the past decade. Resins are the most preferred restorative material. However, their failure rate was observed to be high. This review is aimed for clinician, discussing the influence of human and bacterial enzymes on resin restorations. Composite restoration failure is multifactorial with an interplay of mechanical functions such as masticatory forces and abrasion with biological factors such as host modulated and bacterial enzymes. Salivary esterases and bacterial esterases act on the ester-link bond of resin restoration to form byproducts of methacrylic acid and Bis-hydroxy-propoxy-phenyl-propane. Salivary enzymes form microgaps between the resin-tooth interface and provide a suitable environment for bacterial growth. Bacteria colonize the resin-tooth interface to weaken the resin bond strength. The presence of bacteria draws neutrophils into the hybrid layer. The activation and degranulation of neutrophils leads to enzyme secretions that act on bacteria. However, this can also have adverse effects on resin restoration. Acids prompt the activation of matrix metalloproteinases (MMPs). Proteinases secreted by MMPs uncoil the collagen fibrils of the dentin matrix and degrade tooth structure. The salivary esterases, bacterial esterases, neutrophils, and MMPs work synergistically to degrade dental resin material, resin-tooth interface, and dentin. This causes failure of dental resin restorations and secondary caries formation. Biological degradation of resin restorations is inevitable irrespective of the material and techniques used. Salivary esterases such as cholesterol esterase and pseudocholinesterase and cariogenic bacterial esterase can degrade dental resin, weakening the hybrid layer at the resin-tooth interface, affecting the bond strength, and causing failure. Ester-free resin and incorporation of antimicrobial materials, esterase, and MMP inhibitors are strategies that could ameliorate degradation of the restoration.

The Beneficial and Adverse Effects of Phytoestrogens

The most well-known phytoestrogens (flavonoids, isoflavonoids, lignans, coumestans, stilbenes, and prenylflavonoids) are isoflavonoids, which are important active ingredients in medicinal and food plants. They are highly abundant in the Fabaceae family. More than 1,000 types of isoflavonoids have been isolated from nearly 300 kinds of plants, and more are being discovered through modern analytical methods. Glycosides O and C of isoflavonoids are poorly absorbed in the intestine. They are converted by bacterial esterases and/or β-glycosidase enzymes to aglycones, which are absorbed more efficiently. Their bioavailability shows significant differences due to variation in the intestinal microflora of various races. The compounds formed during their biotransformation are structurally similar to estrogens. In Traditional Chinese medicine, several herbs rich in phytoestrogens are used to prevent and cure various diseases, such as osteoporosis, cardiovascular diseases, diabetes mellitus, hypertension, hyperlipidemia, tumors, and inflammation; additionally, 185 herbs are used to treat menopausal symptoms. Some of these herbs can be used to alleviate the unpleasant symptoms of menopause and treat breast and prostate cancer. From a nutritional physiology perspective, the consumption of Glycine max and Vigna unguiculata should be emphasized. Soy has been consumed in China for about 5,000 years while it was introduced to Europe nearly 300 years ago. Soybean cultivation in Hungary dates back only 100 years. The assessment of the efficacy of phytoestrogens is unclear. Although several experimental and molecular biology studies have shown favorable results, studies on humans have not shown prominent therapeutic benefits. However, comparing and interpreting the findings of modern studies might elucidate the therapeutic utility of phytoestrogens.

Gut commensal Coprococcus comes diminishes the blood pressure‐lowering effect of ester angiotensin‐converting enzyme inhibitors

IntroductionDrug resistant HTN (rHTN) affects around 15% to 20% of hypertensive (HTN) patients. The underlying mechanisms of resistance to treatment remain poorly understood. The majority of angiotensin‐converting enzyme inhibitors (ACEi) are esters, whereby we hypothesized that select gut microbiota hydrolyze ACEi rendering lower efficacy (Figure 1A). To test this hypothesis, we investigated if and which gut microbe modulates the effectiveness of ACEi.MethodsVancomycin, Meropenem and Omeprazole (VMO) were given to 16‐week‐old male Spontaneously Hypertensive Rats (SHR) at 50 mg/kg/day for five days. A single dose of 8mg/kg Quinapril was orally administered to both SHR and SHR+VMO, and blood pressure (BP) was recorded via radio‐telemetry. Liquid chromatography–mass spectrometry was used to measure the catabolism of quinapril. The hydrolysis of p‐nitro‐phenylbutyrate was used to measure the activity of bacterial esterase. 16S rRNA sequencing was used to study the microbial composition. At last, ester ACEi ramipril and non‐ester lisinopril were co‐administered with Coprococcus comes, respectively, to generalize the effect of C. comes on ACEi's effectiveness.ResultsCompared to the SHR, depletion of gut microbiota in the SHR+VMO group preserved the BP lowering effect of Quinapril, an ester ACEi (Figure 1B). The SHR+VMO group showed (1) reduced Coprococcus (Figure 1C); (2) lower esterase activity per gram of cecal microbiota to hydrolyze quinapril (Figure 1D); (3) a 50% lower reduction in quinapril quantity (nmol) after incubation with 1mg of cecal lysate for 3 hr (Figure 1E). C. comes,a species in Coprococcusgenus, catabolized quinapril in vitro and reduced its BP‐lowering effects in the SHR (Figure 2A‐B). Importantly, C. comes also reduced the BP‐lowering effects of ramipril (ester), but not lisinopril (non‐ester) in the SHR (Figure 2C‐D).ConclusionThese observations constitute the first report of an unrecognized role of a select gut microbe, C. comes, in reducing the effectiveness of ester ACEi. As such, this mechanistic study serves as the foundation for expanding clinical management of antihypertensive drug resistance via manipulation of gut microbiota.

The influences of substrates' physical properties on enzymatic PET hydrolysis: Implications for PET hydrolase engineering.

Plastic pollution in diverse terrestrial and marine environments is a widely recognised and growing problem. Bio-recycling and upcycling of plastic waste is a potential solution to plastic pollution, as these processes convert plastic waste into useful materials. Polyethylene terephthalate (PET) is the most abundant plastic waste, and this material can be degraded by a class of recently discovered bacterial esterase enzymes known as PET hydrolases (PETase). Investigations of the enzymatic hydrolysis of diverse PET molecules have clearly revealed that the biodegradability of various PET substrates depends on both their chemical structure and physical properties, including polymer length, crystallinity, glass transition temperature, surface area, and surface charge. This review summarises the known impacts of crystallinity and other physical properties on enzymatic PET hydrolysis.

An Esterase with Increased Acetone Tolerance from Bacillus subtilis E9 over Expressed in E. coli BL21 Using pTac Bs-est Vector.

Bacillus subtilis E9 was identified as a potential strain producing esterase. The gene coding esterase from B. subtilis E9 was amplified using esterase-specific primers and the sequence was translated in silico. The presence of conserved catalytic triad amino acid residues (His-Ser-Asp/Glu) confirmed the functional nature of the esterase enzyme. Docking studies conducted with modeled protein and the ligand p-nitrophenyl acetate showed that the amino acid residues interacting with the ligand were Ser77, His76, and Gly103. The gene coding for esterase from B. subtilis E9 was cloned into an assembled vector having Tac promoter (pTac), pUC origin of replication, Ni-Histidine residues, ampicillin cassette, and T7 terminator using Golden gate DNA assembly method. The generated pTac Bs-est (4598bp) recombinant plasmid was transformed and heterologously expressed in Escherichia coli BL21 (DE3) strain. The tagged recombinant protein was purified to yield 43.4% pure protein with specific activity of 772 U/mg. The purified recombinant protein was subjected to peptide sequencing and the identity was confirmed as esterase by peptide tandem mass fragmentation method using the LC-MS/MS analysis. The purified recombinant esterase was found to be organic solvent stable and tolerant up to 5days retaining around 95% residual activity in 30-90% v/v Acetone. The recombinant esterase expressed in our study was found to exhibit better organic solvent stability and tolerance than compared to the original bacterial esterase from B. subtilis E9, a property which could be explored in the biocatalytic and synthetic transformation reactions for industrial applications.

Utilizing a degradation prediction pathway system to understand how a novel methacrylate derivative polymer with flipped external ester groups retains physico-mechanical properties following esterase exposure

ObjectiveThe region of failure for current methacrylates (i.e. derivatives of acrylates) are ester bond linkages that hydrolyze in the presence of salivary and bacterial esterases that break the polymer network backbone. This effect decreases the mechanical properties of methacrylate-based materials. MethodsThe ethylene glycol dimethacrylate (EGDMA) or novel ethylene glycol ethyl methacrylate (EGEMA) discs were prepared using 40 µL of the curing mixture containing photo/co-initiators for 40 s in a PTFE mold at 1000 mW/cm2. The degree of conversion was used as a quality control measure for the prepared discs, followed by physical, mechanical, and chemical characterization of discs properties before and after cholesterol esterase treatment. ResultsAfter 9 weeks of standardized cholesterol esterase (CEase) exposure, EGDMA discs showed exponential loss of material (p = 0.0296), strength (p = 0.0014) and increased water sorption (p = 0.0002) compared to EGEMA discs. We integrated a degradation prediction pathway system to LC/MS and GC/MS analyses to elucidate the degradation by-products of both EGEMA and EGDMA polymers. GC/MS analysis demonstrated that the esterase catalysis was directed to central polymer backbone breakage, producing ethylene glycol, for EGDMA, and to side chain breakage, producing ethanol, for EGEMA. The flipped external ester group linkage design is attributed to EGEMA showing higher resistance to esterase biodegradation and changes in mechanical and physical properties than EGDMA. SignificanceEGEMA is a potential substitute for common macromer diluents, such as EGDMA, based on its resistance to biodegradation effects. This work inspires the flipped external group design to be applied to analogs of current larger, hydrophobic strength bearing macromers used in future dental material formulations.

Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling.

Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biology. Recently, methodology to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunological fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiology. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ methyl esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the methyl ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biology, offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.

Abstract MP38: Identification Of A Gut Microbe That Attenuates The Blood Pressure Lowering Effect Of ACEi Quinapril

Introduction: Treatment resistant hypertension (rHTN) is present in ~20% of all hypertensive patients. rHTN is critical in African American patients who experience early onset, severe outcomes, and weak responsiveness to angiotensin converting enzyme inhibitor (ACEi). The mechanism for drug resistance is unknown. Gut microbiota harbors biotransformative enzymes such as esterase, which may hydrolyze ACEis, reducing absorption. Our study was to identify microbe responsible for ACEi resistance. Methods: 16-week-old male Spontaneously Hypertensive Rats (SHR) were gavaged with (N=12) or without (N=6) Vancomycin, Meropenem, and Omeprazole (VMO) 50 mg/kg/day for five days to deplete the gut microbiota. A single 8mg/kg dose of quinapril was given to SHR and SHR+VMO before blood pressure (BP) recording via telemetry. Quinapril catabolism was quantified by liquid chromatography-mass spectrometry. Bacterial esterase activity was monitored by hydrolysis of p-nitro-phenylbutyrate. Cecal microbiota was analyzed by 16S rDNA. Fecal microbiota were analyzed by metagenomics in 29 (16 black, 13 white) HTN patients. Results: Quinapril lowered BP more in the SHR+VMO than SHR ( P &lt;0.0001). With a 50% reduction in bacterial 16S copy numbers ( P &lt;0.0001), the SHR+VMO group showed (1) reduced Coprococcus ( P &lt;0.0001); (2) lower esterase activity per gram of cecal microbiota to hydrolyze quinapril ( P =0.0065); (3) a 50% lower reduction in quinapril quantity (nmol) after incubation with 1mg of cecal lysate for 3 hr ( P &lt;0.0001); (4) decreased bacterial genes in KEGG drug metabolism pathway ( P &lt;0.0001). The abundance of Coprococcus positively correlated with genes in drug metabolism ( P &lt;0.0001). Importantly, co-administration of quinapril with C. comes, a species in Coprococcus genus, reduced the BP-lowering effects of quinapril in the SHR ( P &lt;0.0001). Comparison of human microbiota demonstrated a higher abundance of C. comes in the black hypertensives (poor ACEi responder) than the white (ACEi responder) ( P =0.0167). Conclusion: We, for the first time, demonstrate a previously unrecognized role of gut microbes in reducing ACEi effectiveness. This serves a foundation for expanding clinical management of antihypertensive drug resistance via manipulation of gut microbiota.

Structure-guided microbial targeting of antistaphylococcal prodrugs.

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase-specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, ultimately identifying several promoieties likely to be serum esterase-resistant and microbially labile. These studies will enable structure-guided design of antistaphylococcal promoieties and expand the range of molecules to target staphylococcal pathogens.

Isolation, purification and characterization of a novel esterase from camel rumen metagenome

Bacterial esterases are gaining the importance in pharmaceuticals and agrochemical industries due to their excellent biocatalytic properties and a wide range of applications. In the present study, a novel gene encoding an esterase (designated as Est-CR) was identified from shotgun metagenomic sequencing data of camel rumen (Camelus dromedarius) liquor. The open reading frame consisted of 1,224bp, which showed 84.03% sequence identity to Bacteroidales bacterium, corresponding to a protein of 407 amino acids and has a catalytic domain belonging to an esterase. Est-CR belonged to family V with GLSMG domain. The purified enzyme with a molecular mass of 62.64 kDa was checked on SDS-PAGE, and its expression was confirmed by western blotting. The enzyme was active and stable over a broad range of temperature (35–65 °C), displayed the maximum activity at 50 °C and pH 7.0. Individually all metal ions inhibited the enzyme activity, while in combination, K2+, Ca2+, Mg2+ and Mn2+ metal ions enhanced the enzyme activity. The detergents strongly inhibited the activity, while EDTA (10 mM) increased the activity of the Est-CR enzyme. The enzyme showed specificity to short-chain substrates and displayed an optimum activity against butyrate ester. This novel enzyme might serve as a promising candidate to meet some harsh industrial processes enzymatic needs.

Depletion of Esterase‐Harboring Bacteria Increases Antihypertensive Efficacy of ACE Inhibitor Quinapril

Introduction Resistant hypertension (rHTN) is a critical impediment to successful control of high blood pressure (BP) and hypertension-linked pathophysiology in 15-20% of all hypertensive patients. rHTN is of importance in African American patients because they experience early onset, increased prevalence and severe outcomes. The mechanisms for drug resistance in rHTN remain unknown. Eggerthellaceae family harbors biotransformative enzymes including esterase, which influences drug catabolism in the gut prior to absorption. Thus, we hypothesized that an increased abundance of bacteria with catabolic capacity for an antihypertensive drug will decrease its efficacy (Fig. A) and may thereby serve as a novel treatment target for rHTN. Methods Ten human feces (4 controlled HTN (cHTN), 6 rHTN) were analyzed by metatranscriptomics. 16-week old male Spontaneously Hypertensive Rats (SHR) were gavaged with (N=12) or without (N=6) Vancomycin, Meropenem, and Omeprazole (VMO), at 50 mg/kg/day for five days to deplete the gut microbiota. To test the quinapril efficacy, a single 8mg/kg dose was gavaged to both SHR and SHR+VMO before BP recording via telemetry. Quinapril catabolism was quantified by liquid chromatography-mass spectrometry. Bacterial esterase activity was monitored by the hydrolysis of p-nitro-phenylbutyrate. Cecal microbiota was analyzed by 16S rDNA. Pearson r correlation was plotted using GraphPad. Results Patients with rHTN showed ~3-fold more Eggerthellaceae compared to cHTN (p=0.0027, Fig. B). With 50% reduction in bacterial 16S copy numbers (P<0.0001, Fig. C), the SHR+VMO group showed (1) reduced Eggerthellaceae (P<0.0001, Fig. D); (2) lower esterase activity per gram of cecal microbiota to hydrolyze quinapril (p=0.0065, Fig. E); (3) a 50% lower reduction in quinapril quantity (Δquinapril, nmol) after incubation with 1mg of cecal lysate (P<0.0001, Fig. F); (4) decreased bacterial genes in KEGG drug metabolism pathway (P<0.0001, Fig. G). The abundance of Eggerthellaceae positively correlated with Δquinapril (P=0.029, Fig. H) and genes in drug metabolism (P<0.0001, Fig. I). Importantly, administration of quinapril to the SHR+VMO led to lowering of BP, compared to SHR (Fig. J). Conclusion Eggerthellaceae was enriched ~3-fold in rHTN. Using SHR, we showed that depletion of Eggerthellaceae and esterase activity of the gut microbiota by VMO increased the antihypertensive efficacy of quinapril, due to protection of quinapril from catabolism in the gut. Therefore, our study, for the first time, provides evidence for a previously unrecognized role of gut microbiota in rHTN. Innovative approaches can now be considered for rHTN based on strategies to protect antihypertensive drugs from catabolism by gut bacteria before reaching the systemic circulation for improved efficacy.

A Thermophilic Bacterial Esterase for Scavenging Nerve Agents: A Kinetic, Biophysical and Structural Study.

Organophosphorous nerve agents (OPNA) pose an actual and major threat for both military and civilians alike, as an upsurge in their use has been observed in the recent years. Currently available treatments mitigate the effect of the nerve agents, and could be vastly improved by means of scavengers of the nerve agents. Consequently, efforts have been made over the years into investigating enzymes, also known as bioscavengers, which have the potential either to trap or hydrolyze these toxic compounds. We investigated the previously described esterase 2 from Thermogutta terrifontis (TtEst2) as a potential bioscavenger of nerve agents. As such, we assessed its potential against G-agents (tabun, sarin, and cyclosarin), VX, as well as the pesticide paraoxon. We report that TtEst2 is a good bioscavenger of paraoxon and G-agents, but is rather slow at scavenging VX. X-ray crystallography studies showed that TtEst2 forms an irreversible complex with the aforementioned agents, and allowed the identification of amino-acids, whose mutagenesis could lead to better scavenging properties for VX. In conjunction with its cheap production and purification processes, as well as a robust structural backbone, further engineering of TtEst2 could lead to a stopgap bioscavenger useful for in corpo scavenging or skin decontamination.