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Zearalenone and radicicol are resorcylic acid lactones produced by numerous plant pathogenic fungi. Zearalenone is a non-steroidal estrogen mimic that can cause serious reproductive issues in livestock that consume contaminated feed. Radicicol is a potent inhibitor of the molecular chaperone Hsp90, which, in plants, has an important role in coordinating the host's immune response during infection. Here, we describe the identification and characterization of a soil-borne strain of the Gram-positive bacterium Aeromicrobium sp. capable of hydrolyzing the macrolide ring of resorcylic acid lactones, including zearalenone and radicicol. Proteomic analysis of biochemically enriched fractions from the isolated and cultured bacterium identified an α/β-hydrolase responsible for this activity. A recombinantly expressed and purified form of the hydrolase (termed RALH) was active against both zearalenone and radicicol. Interpretation of high-resolution mass spectrometry and NMR data confirmed the structures of the enzymatic products as the previously reported non-toxic metabolite hydrolyzed zearalenone and hydrolyzed radicicol. Hydrolyzed radicicol was demonstrated to no longer inhibit the ATPase activity of the Saccharomyces cerevisiae Hsp90 homolog in vitro. Enzymatic degradation of resorcylic acid lactones will enable insight into their biological functions. [ABSTRACT FROM AUTHOR]

Strigolactone is the collective name for compounds containing a butenolide as a part of their structure, first discovered as compounds that induce seed germination of root parasitic plants. They were later found to be rhizosphere signaling molecules that induce hyphal branching of arbuscular mycorrhizal fungi, and, finally, they emerged as a class of plant hormones. Strigolactones are found in root exudates, where they display a great variability in their chemical structure. Their structure varies among plant species, and multiple strigolactones can exist in one species. Over 30 strigolactones have been identified, yet the chemical structure of the strigolactone that functions as an endogenous hormone and is found in the above-ground parts of plants remains unknown. We discuss our current knowledge of the synthetic pathways of diverse strigolactones and their regulation, as well as recent progress in identifying strigolactones as plant hormones. Strigolactone is perceived by the DWARF14 (D14), receptor, an α/β hydrolase which originated by gene duplication of KARRIKIN INSENSITIVE 2 (KAI2). D14 and KAI2 signaling pathways are partially overlapping paralogous pathways. Progress in understanding the signaling mechanisms mediated by two α/β hydrolase receptors as well as remaining challenges in the field of strigolactone research are reviewed. [ABSTRACT FROM AUTHOR]

Zearalenone and radicicol are resorcylic acid lactones produced by numerous plant pathogenic fungi. Zearalenone is a non-steroidal estrogen mimic that can cause serious reproductive issues in livestock that consume contaminated feed. Radicicol is a potent inhibitor of the molecular chaperone Hsp90, which, in plants, has an important role in coordinating the host’s immune response during infection. Here, we describe the identification and characterization of a soil-borne strain of the Gram-positive bacterium Aeromicrobium sp. capable of hydrolyzing the macrolide ring of resorcylic acid lactones, including zearalenone and radicicol. Proteomic analysis of biochemically enriched fractions from the isolated and cultured bacterium identified an α/β-hydrolase responsible for this activity. A recombinantly expressed and purified form of the hydrolase (termed RALH) was active against both zearalenone and radicicol. Interpretation of high-resolution mass spectrometry and NMR data confirmed the structures of the enzymatic products as the previously reported non-toxic metabolite hydrolyzed zearalenone and hydrolyzed radicicol. Hydrolyzed radicicol was demonstrated to no longer inhibit the ATPase activity of the Saccharomyces cerevisiae Hsp90 homolog in vitro. Enzymatic degradation of resorcylic acid lactones will enable insight into their biological functions.

Strigolactones (SLs) were initially discovered as germination inducers for root parasitic plants. In 2015, three groups independently reported the characterization of the SL receptor in the root parasitic plant Striga hermonthica , which causes significant damage to crop production, particularly in sub-Saharan Africa. The characterized receptors belong to HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE2 (HTL/KAI2), which is a member of the α/β-hydrolase protein superfamily. In non-parasitic plants, HTL/KAI2 perceives the smoke-derived germination inducer karrikin and a yet-unidentified endogenous ligand. However, root parasitic plants evolved a specific clade of HTL/KAI2 that has diverged from the KAI2 clade of non-parasitic plants. The S. hermonthica SL receptors are included in this specific clade, which is called KAI2 divergent (KAI2d). Orobanche minor is an obligate root holoparasitic plant that grows completely dependent on the host for water and nutrients because of a lack of photosynthetic ability. Previous phylogenetic analysis of KAI2 proteins in O. minor has demonstrated the presence of at least five KAI2d clade genes. Here, we report that KAI2d3 and KAI2d4 in O. minor have the ability to act as the SL receptors. They directly interact with SLs in vitro, and when expressed in Arabidopsis , they rescue thermo-inhibited germination in response to the synthetic SL analog GR24. In particular, KAI2d3 showed high sensitivity to GR24 when expressed in Arabidopsis , suggesting that this receptor enables highly sensitive SL recognition in O. minor. Furthermore, we provide evidence that these KAI2d receptors are involved in the perception of sesquiterpene lactones, non-strigolactone-type germination inducers. [ABSTRACT FROM AUTHOR]

DWARF14 (D14) and HTL/KAI2 (KAI2) are paralogous receptors in the α/β-hydrolase superfamily. D14 is the receptor for a class of plant hormones, strigolactones (SLs), and KAI2 is the receptor for the smoke-derived seed germination inducer, Karrikin (KAR), in Arabidopsis. Germinone (Ger) was previously reported as a KAI2 agonist with germination-inducing activity for thermo-inhibited Arabidopsis seed. However, Ger was not specific to KAI2, and could also bind to D14. It was reported that SL analogs with a desmethyl-type D-ring structure are specifically recognized by KAI2. On the basis of this observation, we synthesized a desmethyl-type germinone (dMGer). We found that dMGer is highly specific to KAI2. Moreover, dMGer induced Arabidopsis seed germination more effectively than did Ger. In addition, dMGer induced the seed germination of Arabidopsis in a manner independently of GA, a well-known germination inducer in plants. [Display omitted] • desmethylGerminone (dMGer) specifically interacts with the HTL/KAI2 receptor. • dMGer induces thermo-inhibited germination of Arabidopsis. • dMGer induces Arabidopsis germination ia a gibberellin-independent manner. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel hyperstable lipase protein (TpS9) is over-expressed in a mesophilic host. • TpS9 has a well-conserved penta-peptide (GLSAG) motif and α/β-hydrolase fold. • TpS9 production is enhanced by 9.02-fold using IPTG-independent auto-induction. • TpS9 revealed thermal (30–85 °C) and pH (6.0–9.0) stability for 8 h. • Compatible and resistant towards various commercial detergents and modulators. • Act as laundry cleaning bio-additive and degrade animal fat waste. A peptidase S9 prolyl oligopeptidase domain from Thermotoga petrophila RKU-1 T (TpS9) was over-expressed as an active, soluble and hyperstable lipolytic enzyme in the mesophilic host system. The sequence analysis demonstrated, TpS9 is an esterase/lipase-like protein belongs to alpha/beta (α/β)-hydrolase superfamily with a well-conserved penta-peptide (GLSAG) motif and α/β-hydrolase fold. Various approaches (induction and cultivation) were employed to enrich TpS9 production, 6.04- and 7.26-fold increment was observed with IPTG (0.4 mM) and lactose (200 mM) in the modified 4ZB medium (pH 7.0), but with IPTG-independent auto-induction strategy 9.02-fold augmentation was achieved after 16 h incubation at 24 °C (150 rev min−1). Purified TpS9 showed optimal activity in McIlvaine buffer (pH 6.5) at 80–85 °C, and revealed great thermal (30–85 °C) and pH (6.0–9.0) for 8 h. No obvious constraint was perceived with various metal ions, surfactants, commercial laundry detergents, and chemical modulators. Whereas, TpS9 activity was improved with Ca2+, Mn2+, and Mg2+ by 210 %, 142.5 %, and 134.3 %, respectively. With 2.5 M NaCl (215 %), 50 % (v/v) methanol (140 %), 50 % (v/v) ethanol (126.6 %), 50 % (v/v) n -butanol (122.3 %), 50 % (v/v) isopropanol (120.4 %), 50 % (v/v) acetone (118.6 %) and 50 % (v/v) glycerol (113.2 %) TpS9 activity was also enriched. TpS9 demonstrated great affinity toward natural oils and p -nitrophenyl ester substrates, but showed peak activity with p -nitrophenyl palmitate (3160 U mg−1). K m , V max , k cat , V max K m -1 and k cat K m -1 of TpS9 with p NPP were 0.421 mM, 4015 µmol mg−1 min−1, 906.4 s−1, 9536.8 min−1, and 2152.96 mM−1 s−1, respectively. Moreover, TPS9 has notable ability to clean stains (5 min) and degrade the animals' fat (3 h). Hence, TpS9 is a favorable candidate as cleaning bio-additive in detergent formulation, fat degradation and various other applications. [ABSTRACT FROM AUTHOR]

Summary: Strigolactones (SLs) are plant hormones and important signalling molecules required to promote arbuscular mycorrhizal (AM) symbiosis. While in plants an α/β‐hydrolase, DWARF14 (D14), was shown to act as a receptor that binds and cleaves SLs, the fungal receptor for SLs is unknown.Since AM fungi are currently not genetically tractable, in this study, we used the fungal pathogen Cryphonectria parasitica, for which gene deletion protocols exist, as a model, as we have previously shown that it responds to SLs. By means of computational, biochemical and genetic analyses, we identified a D14 structural homologue, CpD14.Molecular homology modelling and docking support the prediction that CpD14 interacts with and hydrolyses SLs. The recombinant CpD14 protein shows α/β hydrolytic activity in vitro against the SLs synthetic analogue GR24; its enzymatic activity requires an intact Ser/His/Asp catalytic triad. CpD14 expression in the d14‐1 loss‐of‐function Arabidopsis thaliana line did not rescue the plant mutant phenotype. However, gene inactivation by knockout homologous recombination reduced fungal sensitivity to SLs.These results indicate that CpD14 is involved in SLs responses in C. parasitica and strengthen the role of SLs as multifunctional molecules acting in plant–microbe interactions. [ABSTRACT FROM AUTHOR]

Abstract Unlike the successful immunization of native H. contortus antigens that contributed to the realization of the first commercial vaccine Barbervax, not many studies revealed the encouraging protective efficacies of recombinant H. contortus antigens in laboratory trials or under field conditions. In our preliminary study, H. contortus α/β-hydrolase domain protein (HcABHD) was demonstrated to be an immunomodulatory excretory–secretory (ES) protein that interacts with goat T cells. We herein evaluated the protective capacities of two HcABHD preparations, recombinant HcABHD (rHcABHD) antigen and anti-rHcABHD IgG, against H. contortus challenge via active and passive immunization trials, respectively. Parasitological parameter, antibody responses, hematological pathology and cytokine profiling in unchallenged and challenged goats were monitored and determined throughout both trials. Subcutaneous administration of rHcABHD with Freund adjuvants elicited protective immune responses in challenged goats, diminishing cumulative fecal egg counts (FEC) and total worm burden by 54.0% and 74.2%, respectively, whereas passive immunization with anti-rHcABHD IgG conferred substantial protection to challenged goats by generating a 51.5% reduction of cumulative FEC and a 73.8% reduction of total worm burden. Additionally, comparable changes of mucosal IgA levels, circulating IgG levels, hemoglobin levels, and serum interleukin (IL)-4 and IL-17A levels were observed in rHcABHD protein/anti-rHcABHD IgG immunized goats in both trials. Taken together, the recombinant version of HcABHD might have further application under field conditions in protecting goats against H. contortus infection, and the integrated immunological pipeline of ES antigen identification, screening and characterization may provide new clues for further development of recombinant subunit vaccines to control H. contortus.

SUMMARY: KARRIKIN INSENSITIVE2 (KAI2) is an α/β‐hydrolase required for plant responses to karrikins, which are abiotic butenolides that can influence seed germination and seedling growth. Although represented by four angiosperm species, loss‐of‐function kai2 mutants are phenotypically inconsistent and incompletely characterised, resulting in uncertainties about the core functions of KAI2 in plant development. Here we characterised the developmental functions of KAI2 in the grass Brachypodium distachyon using molecular, physiological and biochemical approaches. Bdkai2 mutants exhibit increased internode elongation and reduced leaf chlorophyll levels, but only a modest increase in water loss from detached leaves. Bdkai2 shows increased numbers of lateral roots and reduced root hair growth, and fails to support normal root colonisation by arbuscular‐mycorrhizal (AM) fungi. The karrikins KAR1 and KAR2, and the strigolactone (SL) analogue rac‐GR24, each elicit overlapping but distinct changes to the shoot transcriptome via BdKAI2. Finally, we show that BdKAI2 exhibits a clear ligand preference for desmethyl butenolides and weak responses to methyl‐substituted SL analogues such as GR24. Our findings suggest that KAI2 has multiple roles in shoot development, root system development and transcriptional regulation in grasses. Although KAI2‐dependent AM symbiosis is likely conserved within monocots, the magnitude of the effect of KAI2 on water relations may vary across angiosperms. Significance Statement: KAI2 is an α/β‐hydrolase receptor for abiotic karrikins and yet elusive plant hormones. The roles of KAI2 have been extensively studied in the dicotyledon Arabidopsis. Here we provide a comprehensive characterisation of the roles of KAI2 in the monocotyledon Brachypodium distachyon. [ABSTRACT FROM AUTHOR]

Summary: Strigolactones and karrikins are butenolide molecules that regulate plant growth. They are perceived by the α/β‐hydrolase DWARF14 (D14) and its homologue KARRIKIN INSENSITIVE2 (KAI2), respectively. Plant‐derived strigolactones have a butenolide ring with a methyl group that is essential for bioactivity. By contrast, karrikins are abiotic in origin, and the butenolide methyl group is nonessential. KAI2 is probably a receptor for an endogenous butenolide, but the identity of this compound remains unknown.Here we characterise the specificity of KAI2 towards differing butenolide ligands using genetic and biochemical approaches.We find that KAI2 proteins from multiple species are most sensitive to desmethyl butenolides that lack a methyl group. Desmethyl‐GR24 and desmethyl‐CN‐debranone are active by KAI2 but not D14. They are more potent KAI2 agonists compared with their methyl‐substituted reference compounds both in vitro and in plants. The preference of KAI2 for desmethyl butenolides is conserved in Selaginella moellendorffii and Marchantia polymorpha, suggesting that it is an ancient trait in land plant evolution.Our findings provide insight into the mechanistic basis for differential ligand perception by KAI2 and D14, and support the view that the endogenous substrates for KAI2 and D14 have distinct chemical structures and biosynthetic origins. [ABSTRACT FROM AUTHOR]

Unlike the successful immunization of native H. contortus antigens that contributed to the realization of the first commercial vaccine Barbervax, not many studies revealed the encouraging protective efficacies of recombinant H. contortus antigens in laboratory trials or under field conditions. In our preliminary study, H. contortus α/β-hydrolase domain protein (HcABHD) was demonstrated to be an immunomodulatory excretory–secretory (ES) protein that interacts with goat T cells. We herein evaluated the protective capacities of two HcABHD preparations, recombinant HcABHD (rHcABHD) antigen and anti-rHcABHD IgG, against H. contortus challenge via active and passive immunization trials, respectively. Parasitological parameter, antibody responses, hematological pathology and cytokine profiling in unchallenged and challenged goats were monitored and determined throughout both trials. Subcutaneous administration of rHcABHD with Freund adjuvants elicited protective immune responses in challenged goats, diminishing cumulative fecal egg counts (FEC) and total worm burden by 54.0% and 74.2%, respectively, whereas passive immunization with anti-rHcABHD IgG conferred substantial protection to challenged goats by generating a 51.5% reduction of cumulative FEC and a 73.8% reduction of total worm burden. Additionally, comparable changes of mucosal IgA levels, circulating IgG levels, hemoglobin levels, and serum interleukin (IL)-4 and IL-17A levels were observed in rHcABHD protein/anti-rHcABHD IgG immunized goats in both trials. Taken together, the recombinant version of HcABHD might have further application under field conditions in protecting goats against H. contortus infection, and the integrated immunological pipeline of ES antigen identification, screening and characterization may provide new clues for further development of recombinant subunit vaccines to control H. contortus. [ABSTRACT FROM AUTHOR]

Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment in vitro, accepting a wide range of alcohols (C2–C10) but only a small range of acyl donors (C2–C4). We show that alcoholysis occurs in vivo in several Crabtree negative yeast species but also in engineered Saccharomyces cerevisiae strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters in vivo by overexpressing Eat1 in Clostridium beijerinckii. Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the in vitro alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products.

The α/β-hydrolase domain (ABHD) proteins belonging to α/β-hydrolase (ABH) superfamily are ubiquitously distributed throughout all the organisms, and their functional roles have been implicated in energy metabolism, cell signaling, growth and development. In our preliminary work, we identified a novel ABHD protein derived from Haemonchus contortus excretory-secretory (ES) proteins (HcESPs) that interacted with host T cells. Here, we demonstrated that H. contortus ABHD (HcABHD) protein, expressed in all life-cycle stages of H. contortus, is a mammalian ABHD17 homolog with immunodiagnostic utility and lipase activity. Given its catalytic activities and immunomodulatory potentials, we further investigated the functional diversity of HcABHD as an individual ES protein in parasite-host interactions. HcABHD protein may serve as depalmitoylase or thioesterase to suppress cell viability, inhibit cell proliferation, induce intrinsic and extrinsic T cell apoptosis, and cause cell cycle arrested at G1 phase. Moreover, recombinant HcABHD stimuli exerted critical controls on T cell cytokine production profiles, predominantly by inhibiting the secretions of interleukin (IL)-4, interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-β) 1, and promoting IL-10 production. As the immunomodulator acting at the parasite-host interface, HcABHD protein may have potential applications for the vaccine development of therapeutic intervention. Together, these findings may help illuminate the molecular and particularly immunomodulatory aspects of ES proteins and contribute to an enhanced understanding of parasite immune evasion in H. contortus-host biology.

Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment in vitro , accepting a wide range of alcohols (C2–C10) but only a small range of acyl donors (C2–C4). We show that alcoholysis occurs in vivo in several Crabtree negative yeast species but also in engineered Saccharomyces cerevisiae strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters in vivo by overexpressing Eat1 in Clostridium beijerinckii. Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the in vitro alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products. [ABSTRACT FROM AUTHOR]

The α/β-hydrolase domain (ABHD) proteins belonging to α/β-hydrolase (ABH) superfamily are ubiquitously distributed throughout all the organisms, and their functional roles have been implicated in energy metabolism, cell signaling, growth and development. In our preliminary work, we identified a novel ABHD protein derived from Haemonchus contortus excretory-secretory (ES) proteins (HcESPs) that interacted with host T cells. Here, we demonstrated that H. contortus ABHD (HcABHD) protein, expressed in all life-cycle stages of H. contortus , is a mammalian ABHD17 homolog with immunodiagnostic utility and lipase activity. Given its catalytic activities and immunomodulatory potentials, we further investigated the functional diversity of HcABHD as an individual ES protein in parasite-host interactions. HcABHD protein may serve as depalmitoylase or thioesterase to suppress cell viability, inhibit cell proliferation, induce intrinsic and extrinsic T cell apoptosis, and cause cell cycle arrested at G1 phase. Moreover, recombinant HcABHD stimuli exerted critical controls on T cell cytokine production profiles, predominantly by inhibiting the secretions of interleukin (IL)-4, interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-β) 1, and promoting IL-10 production. As the immunomodulator acting at the parasite-host interface, HcABHD protein may have potential applications for the vaccine development of therapeutic intervention. Together, these findings may help illuminate the molecular and particularly immunomodulatory aspects of ES proteins and contribute to an enhanced understanding of parasite immune evasion in H. contortus -host biology. [ABSTRACT FROM AUTHOR]

Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/β-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates. [ABSTRACT FROM AUTHOR]

Cryptosporidium parvum is a globally important zoonotic parasite capable of causing severe to deadly diarrhea in humans and animals. Its small genome (~9.1 Mb) encodes not only a highly streamlined metabolism, but also a 25-kb, 3-module fatty acid synthase (CpFAS1) and a 40-kb, 7-module polyketide synthase (CpPKS1). The two megasynthases contain a C-terminal reductase domain to release the final products with predicted chain lengths of ≥C22 for CpFAS1 or C28 to C38 for CpPKS1.The parasite genome also encodes a discrete thioesterase ortholog, suggesting its role to be an alternative tool in releasing the final products from CpFAS1 and/or CpPKS1, or as an editor to remove non-reactive residues or aberrant intermediates, or to control starter units as seen in other parasites. In this study, we have confirmed that this C. parvum thioesterase is a type II thioesterase (thus named as CpTEII). CpTEII contains motifs and a catalytic triad characteristic to the type II thioesterase family. CpTEII is expressed during the entire parasite life cycle stages with the highest levels of expression in the later developmental stages. CpTEII showed the highest hydrolytic activity toward C10:0 decanoyl-CoA, so we speculated that CpTEII may mainly act as an editor to remove non-reactive residues and/or aberrant medium acyl chain from CpFAS1 and/or CpPKS1. However, we cannot rule out the possibility that CpTEII may also participate in the release of final products from CpFAS1 because of its moderate activity on C20:0, C:22:0 and C24:0 acyl-CoA thioesters (i.e., ~20–30% activity vs. decanoyl-CoA).

Main conclusion: Two newly identified phytohormone cleaving esterases from Olea europaea are responsible for the glucosidase-initiated activation of the specialized metabolites ligstroside and oleuropein. Biosynthetic routes leading to the formation of plant natural products are tightly orchestrated enzymatic sequences usually involving numerous specialized catalysts. After their accumulation in plant cells and tissues, otherwise non-reactive compounds can be enzymatically activated, e.g., in response to environmental threats, like pathogen attack. In olive (Olea europaea), secoiridoid-derived phenolics, such as oleuropein or ligstroside, can be converted by glucosidases and as yet unidentified esterases to oleoside aldehydes. These are not only involved in pathogen defense, but also bear considerable promise as pharmaceuticals or neutraceuticals. Making use of the available olive genomic data, we have identified four novel methylesterases that showed significant homology to the polyneuridine aldehyde esterase (PNAE) from Rauvolfia serpentina, an enzyme acting on a distantly related metabolite group (monoterpenoid indole alkaloids, MIAs) also featuring a secoiridoid structural component. The four olive enzymes belong to the α/ß-hydrolase fold family and showed variable in vitro activity against methyl esters of selected plant hormones, namely jasmonic acid (MeJA), indole acetic acid (MeIAA), as well as salicylic acid (MeSA). None of the identified catalysts were directly active against the olive metabolites oleuropein, ligstroside, or oleoside 11-methyl ester. When employed in a sequential reaction with an appropriate glucosidase, however, two were capable of hydrolyzing these specialized compounds yielding reactive dialdehydes. This suggests that the esterases play a pivotal role in the activation of the olive secoiridoid polyphenols. Finally, we show that several of the investigated methylesterases exhibit a concomitant in vitro transesterification capacity—a novel feature, yielding ethyl esters of jasmonic acid (JA) or indole-3-acetic acid (IAA). [ABSTRACT FROM AUTHOR]

Cryptosporidium parvum is a globally important zoonotic parasite capable of causing severe to deadly diarrhea in humans and animals. Its small genome (~9.1 Mb) encodes not only a highly streamlined metabolism, but also a 25-kb, 3-module fatty acid synthase (CpFAS1) and a 40-kb, 7-module polyketide synthase (CpPKS1). The two megasynthases contain a C-terminal reductase domain to release the final products with predicted chain lengths of ≥C22 for CpFAS1 or C28 to C38 for CpPKS1.The parasite genome also encodes a discrete thioesterase ortholog, suggesting its role to be an alternative tool in releasing the final products from CpFAS1 and/or CpPKS1, or as an editor to remove non-reactive residues or aberrant intermediates, or to control starter units as seen in other parasites. In this study, we have confirmed that this C. parvum thioesterase is a type II thioesterase (thus named as CpTEII). CpTEII contains motifs and a catalytic triad characteristic to the type II thioesterase family. CpTEII is expressed during the entire parasite life cycle stages with the highest levels of expression in the later developmental stages. CpTEII showed the highest hydrolytic activity toward C10:0 decanoyl-CoA, so we speculated that CpTEII may mainly act as an editor to remove non-reactive residues and/or aberrant medium acyl chain from CpFAS1 and/or CpPKS1. However, we cannot rule out the possibility that CpTEII may also participate in the release of final products from CpFAS1 because of its moderate activity on C20:0, C:22:0 and C24:0 acyl-CoA thioesters (i.e., ~20–30% activity vs. decanoyl-CoA). [ABSTRACT FROM AUTHOR]

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD‐I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure–function relationships of cold‐adapted enzymes. [ABSTRACT FROM AUTHOR]

The endocannabinoid (eCB) system is an important part of both the human central nervous system (CNS) and peripheral tissues. It is involved in the regulation of various physiological and neuronal processes and has been associated with various diseases. The eCB system is a complex network composed of receptor molecules, their cannabinoid ligands, and enzymes regulating the synthesis, release, uptake, and degradation of the signalling molecules. Although the eCB system and the molecular processes of eCB signalling have been studied extensively over the past decades, the involved molecules and underlying signalling mechanisms have not been described in full detail. An example pose the two poorly characterised eCB-degrading enzymes α/β-hydrolase domain protein six (ABHD6) and ABHD12, which have been shown to hydrolyse 2-arachidonoyl glycerol—the main eCB in the CNS. We review the current knowledge about the eCB system and the role of ABHD6 and ABHD12 within this important signalling system and associated diseases. Homology modelling and multiple sequence alignments highlight the structural features of the studied enzymes and their similarities, as well as the structural basis of disease-related ABHD12 mutations. However, homologies within the ABHD family are very low, and even the closest homologues have widely varying substrate preferences. Detailed experimental analyses at the molecular level will be necessary to understand these important enzymes in full detail. [ABSTRACT FROM AUTHOR]

Strigolactones (SLs) are a class of terpenoid lactones initially identified as seed germination stimulants for root parasitic plants more than 50 years ago. Long after this initial discovery, SLs were re-characterized as the symbiotic signals for arbuscular mycorrhizal fungi that supply inorganic nutrients, such as phosphate, to their host plants. In 2008, SLs were found to be endogenous plant hormones that regulate shoot branching in plants. The discovery of SLs as a new class of plant hormones has significantly advanced research in this field. Studies over the past 15 years have elucidated almost the entire pathway of SL biosynthesis and the overall mechanism of its signaling. This review summarizes research on the SL biosynthetic pathway, and the current state of knowledge of the SL perception mechanism., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The use of cannabis is a well-established component risk factor for schizophrenia, particularly in adolescent individuals with genetic predisposition for the disorder. Alterations to the endocannabinoid system have been found in the prefrontal cortex of patients with schizophrenia. Thus, we assessed whether molecular alterations exist in the endocannabinoid signalling pathway during brain development in a mouse model for the schizophrenia risk gene neuregulin 1 (Nrg1). We analysed transcripts encoding key molecules of the endocannabinoid system in heterozygous transmembrane domain Nrg1 mutant mice (Nrg1 TM HET), which is known to have increased sensitivity to cannabis exposure. Tissue from the prelimbic cortex and hippocampus of male and female Nrg1 TM HET mice and wild type-like littermates was collected at postnatal days (PNDs) 7, 10, 14, 21, 28, 35, 49, and 161. Quantitative polymerase chain reaction was conducted to assess mRNA levels of cannabinoid receptor 1 (CB1R) and enzymes for the synthesis and breakdown of the endocannabinoid 2-arachidonoylglycerol [i.e., diacylglycerol lipase alpha (DAGLα), monoglyceride lipase (MGLL), and α/β-hydrolase domain-containing 6 (ABHD6)]. No sex differences were found for any transcripts in either brain region; thus, male and female data were pooled. Hippocampal and cortical mRNA expression of DAGLα, MGLL, and ABHD6 increased until PND 21-35 and then decreased and stabilised for the rest of postnatal development. Hippocampal CB1R mRNA expression increased until PND 21 and decreased after this age. Expression levels of these endocannabinoid markers did not differ in Nrg1 TM HET compared to control mice at any time point. Here, we demonstrate dynamic changes in the developmental trajectory of several key endocannabinoid system transcripts in the mouse brain, which may correspond with periods of endocannabinoid system maturation. Nrg1 TM HET mutation did not alter the developmental trajectory of the endocannabinoid markers assessed, suggesting that other mechanisms may be responsible for the exaggerated cannabinoid susceptibility in these mice. [ABSTRACT FROM AUTHOR]

The opportunistic pathogen Pseudomonas aeruginosa colonizes the lungs of susceptible individuals by deploying virulence factors targeting host defenses. The secreted factor Cif (cystic fibrosis transmembrane conductance regulator inhibitory factor) dysregulates the endocytic recycling of CFTR and thus reduces CFTR abundance in host epithelial membranes. We have postulated that the decrease in ion secretion mediated by Cif would slow mucociliary transport and decrease bacterial clearance from the lungs. To test this hypothesis, we explored the effects of Cif in cultured epithelia and in the lungs of mice. We developed a strategy to interpret the "hurricane-like" motions observed in reconstituted cultures and identified a Cif-mediated decrease in the velocity of mucus transport in vitro. Presence of Cif also increased the number of bacteria recovered at two time points in an acute mouse model of pneumonia caused by P. aeruginosa. Furthermore, recent work has demonstrated an inverse correlation between the airway concentrations of Cif and 15-epi-lipoxin A4, a proresolving lipid mediator important in host defense and the resolution of pathogen-initiated inflammation. Here, we observe elevated levels of 15-epi-lipoxin A4 in the lungs of mice infected with a strain of P. aeruginosa that expresses only an inactive form of cif compared with those mice infected with wild-type P. aeruginosa. Together these data support the inclusion of Cif on the list of virulence factors that assist P. aeruginosa in colonizing and damaging the airways of compromised patients. Furthermore, this study establishes techniques that enable our groups to explore the underlying mechanisms of Cif effects during respiratory infection. [ABSTRACT FROM AUTHOR]

Microsporidia are a group of widespread fungi-related obligate intracellular parasites. Direct contact of most microsporidia with the cytoplasm of an infected host cell entails possible secretion of various proteins from the parasite that allows control physiological processes of the host. Earlier, by means of polyclonal antibodies against α/β-hydrolase of microsporidium Paranosema locustae, the secretion of large amounts of the enzyme into the cytoplasm of fat body cells of infected migratory locust Locusta migratoria was demonstrated. However, yeast fungi Pichia pastoris did not recognize this enzyme as a secretory one during its heterologous expression. In the present study, a library of recombinant single-chain antibodies (scFv fragments) against proteins of the infected fat body of locust was constructed. The use of the phage display technology enabled choosing a miniantibody that specifically recognized the studied enzyme. Immunoblotting and immunolabeling of frozen sections of locust fat body with the selected scFv fragment confirmed the fact of secretion of P. locustae α/β-hydrolase (as two forms of different size) into the infected host cell. Prospects of using the selected scFv fragment for further studies of the secretion mechanism of the parasite's protein and its role in host-parasite interactions are discussed. [ABSTRACT FROM AUTHOR]

A novel lipolytic enzyme-producing endophytic strain PC2 was successfully isolated from the seeds of an ideal bioenergy plant Pistacia chinensis Bunge. Based on the analysis of morphology and 16S rRNA sequence, bacterial strain PC2 was identified as a subspecies of Pseudomonas putida, therefore named as P. putida PC2. Whole-genome sequencing showed PC2 contained a 1224-nucleotide lipase gene (named lip-PC2) predicted to encode a 407-amino-acid protein. Purified lipases from both the original PC2 strain and heterologously expressed Escherichia coli were nearly 50 kD with specific activity of 9.48 U/mL. LIP-PC2 displayed the maximal activity at 50°C or pH 8.0, and maintained above 80% relative activity in the range of from 40 to 60°C or pH in the range of from 6.0 to 8.0, indicating thermostable and alkaline properties. Enzyme activity was enhanced by Mg, Na and Mn, but strongly inhibited by Cu, Zn Co, EDTA as well as organic solvents and surfactants. Additionally, the analysis of amino acid sequence and structure indicated that LIP-PC2 was a novel member belonging to family I.3 of bacterial lipolytic enzymes and its catalytic triad was consisted of Ser-200, Asp-342 and His-374. [ABSTRACT FROM AUTHOR]

Plants have evolved secondary metabolite biosynthetic pathways of immense rich diversity. The genes encoding enzymes for secondary metabolite biosynthesis have evolved through gene duplication followed by neofunctionalization, thereby generating functional diversity. Emerging evidence demonstrates that some of those enzymes catalyze reactions entirely different from those usually catalyzed by other members of the same family; e.g. transacylation catalyzed by an enzyme similar to a hydrolytic enzyme. Tuliposide-converting enzyme (TCE), which we recently discovered from tulip, catalyzes the conversion of major defensive secondary metabolites, tuliposides, to antimicrobial tulipalins. The TCEs belong to the carboxylesterase family in the α/β-hydrolase fold superfamily, and specifically catalyze intramolecular transesterification, but not hydrolysis. This non-ester-hydrolyzing carboxylesterase is an example of an enzyme showing catalytic properties that are unpredictable from its primary structure. This review describes the biochemical and physiological aspects of tulipalin biogenesis, and the diverse functions of plant carboxylesterases in the α/β-hydrolase fold superfamily. Both canonical ester-hydrolyzing and atypical non-ester hydrolyzing functions of carboxylesterases are required for secondary metabolism in plants. [ABSTRACT FROM PUBLISHER]

Circular permutation (CP) involves the cleavage of polypeptide to obtain new termini, which can result in different protein structures and functions. It has been demonstrated to be an effective strategy for the evolution of proteins, but the lack of principle for selecting CP site to construct functional variants is still a challenge for CP. In this study we performed the CP analysis of the typical esterase Rh Est1 to explore the CP site-selection strategy of the α/β-hydrolase fold family. A CP library of 97 mutants was generated to identify the effect of CP on three characteristic regions of Rh Est1 including the flexible cap domain (Region 1), the region around the entrance to substrate binding pocket (Region 2) and the surface-exposed sectors in catalytic domain (Region 3). We found the protein folding, stability and bioactivity of CP variants were altered significantly and the CP sites of active variants were mainly located in the flexible loops. These studies reveal the importance of site-selection for CP and provide more information for CP of other α/β-hydrolases. [ABSTRACT FROM AUTHOR]

Our understanding of the genes involved in Alzheimer's disease (AD) is incomplete. Using subtractive cloning technology, we discovered that the α/β-hydrolase fold protein gene NDRG2 (NDRG family member 2) is upregulated at both the RNA and protein levels in AD brains. Expression of NDRG2 in affected brains was revealed in (1) cortical pyramidal neurons, (2) senile plaques and (3) cellular processes of dystrophic neurons. Overexpression of two splice variants encoding a long and short NDRG2 isoform in hippocampal pyramidal neurons of transgenic mice resulted in localization of both isoforms to dendritic processes. Taken together, our findings suggest that NDRG2 upregulation is associated with disease pathogenesis in the human brain and provide new insight into the molecular changes that occur in AD.

Homoserine O-acetyltransferase (HTA) catalyzes the formation of L- O-acetyl-homoserine from L-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus ( SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/β-hydrolase superfamily, consisting of two distinct domains: a core α/β-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA. [ABSTRACT FROM AUTHOR]

EstN2 is a novel α/β-hydrolase originating from the ammonia-oxidizing thaumarchaeon Candidatus Nitrososphaera gargensis. The genome of the organism was sequenced and genes conferring putative lipolytic activity were amplified and cloned into Escherichia coli as a heterologous host. Through function-based screening, esterase and lipase activity was detected. A recombinant enzyme designated EstN2 was successfully expressed, purified and crystallized. The crystals belonged to space group I2, with one molecule per asymmetric unit, and diffracted X-rays to 1.5 Å resolution. [ABSTRACT FROM AUTHOR]

The neuroligins are cell adhesion proteins whose extracellular domain belongs to the α/β-hydrolase fold family of proteins, mainly containing enzymes and exemplified by acetylcholinesterase. The ectodomain of postsynaptic neuroligins interacts through a calcium ion with the ectodomain of presynaptic neurexins to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic differentiation, maturation, and maintenance, is regulated by gene selection, alternative mRNA splicing, and posttranslational modifications. Mutations leading to deficiencies in the expression, folding, maturation, and binding properties of either partner are associated with autism spectrum disorders. The currently available structural and functional data illustrate how these two families of cell adhesion molecules bridge the synaptic cleft to participate in synapse plasticity and support its dynamic nature. Neuroligin partners distinct from the neurexins, and which may undergo either trans or cis interaction, have also been described, and tridimensional structures of some of them are available. Our study emphasizes the partnership versatility of the neuroligin ectodomain associated with molecular flexibility and alternative binding sites, proposes homology models of the structurally non-characterized neuroligin partners, and exemplifies the large structural variability at the surface of the α/β-hydrolase fold subunit. This study also provides new insights into possible surface binding sites associated with non-catalytic properties of the acetylcholinesterase subunit. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and biofilm-mediated multidrug resistance. It produces and secretes various toxins with hydrolytic activities including phospholipases. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here, we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. At reaction equilibrium, PaPlaB purified from detergent-solubilized membranes of E. coli released fatty acids (FAs) from sn -1 and sn -2 positions of phospholipids at the molar ratio of 51:49. PaPlaB in vitro hydrolyzed P. aeruginosa phospholipids reconstituted in detergent micelles and phospholipids reconstituted in vesicles. Cellular localization studies indicate that PaPlaB is a cell-bound PLA of P. aeruginosa and that it is peripherally bound to both membranes in E. coli , yet the active form was predominantly associated with the cytoplasmic membrane of E. coli. Decreasing the concentration of purified and detergent-stabilized PaPlaB leads to increased enzymatic activity, and at the same time triggers oligomer dissociation. We showed that the free FA profile, biofilm amount and architecture of the wild type and Δ plaB differ. However, it remains to be established how the PLB activity of PaPlaB is regulated by homooligomerisation and how it relates to the phenotype of the P. aeruginosa Δ plaB. This novel putative virulence factor contributes to our understanding of phospholipid degrading enzymes and might provide a target for new therapeutics against P. aeruginosa biofilms. • PaPlaB is a novel phospholipase B of Pseudomonas aeruginosa. • PaPlaB hydrolyses endogenous P. aeruginosa phospholipids. • The activity of PaPlaB is inhibited by oligomerisation. • PaPlaB affects biofilm formation and architecture. [ABSTRACT FROM AUTHOR]

ABSTRACT Tannin acylhydrolase (EC 3.1.1.20) referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannins to release gallic acid. Although the enzyme is useful for various industries, the tertiary structure is not yet determined. In this study, we determined the crystal structure of tannase produced by Lactobacillus plantarum. The tannase structure belongs to a member of α/β-hydrolase superfamily with an additional 'lid' domain. A glycerol molecule derived from cryoprotectant solution was accommodated into the tannase active site. The binding manner of glycerol to tannase seems to be similar to that of the galloyl moiety in the substrate. Proteins 2013; 81:2052-2058. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Highlights: [•] All eukaryotes have at least a single copy of a bem46 ortholog. [•] The catalytic triad of BEM46 is illustrated using sequence and structural analysis. [•] We identified indels in the conserved domain of BEM46 protein. [•] Localization studies of BEM46 protein were carried out using GFP-fusion tagging. [ABSTRACT FROM AUTHOR]

Abstract: Honey bees undergo a physiological transition from nursing to foraging approximately 21 days after adult emergence. This transition is delayed by ethyl oleate (EO), a primer pheromone produced by foragers when exposed to ethanol from fermented nectar. We demonstrate here that two secreted α/β-hydrolases (BeeBase ID: GB11403 and GB13365) are responsible for the reversible esterification of ethanol with oleic acid, giving EO. Expression of hydrolase GB11403 was shown to be significantly up-regulated in foragers, relative to nurses. Tissue perfusion experiments with labeled substrates consistently localized the highest level of EO production in the head, whereas in situ imaging revealed expression of relevant EO biosynthetic genes and enzymatic activity along the esophagus, the site of ethanol exposure during nectar intake. Both α/β-hydrolases were expressed in Pichia pastoris, purified and were shown produce EO in vitro. Experiments with live bees fed ethanol demonstrated that EO formed in regurgitate accumulates in the honey crop and exudes to the exoskeleton, from where it exerts its primer effect on younger bees. [Copyright &y& Elsevier]

Abstract: The neuroligins are postsynaptic cell adhesion proteins whose extracellular domain belongs to the α/β-hydrolase fold family of proteins, a family characterized through the enzyme acetylcholinesterase (AChE) and other enzymes with various substrate specificities. Neuroligin associations with the pre-synaptic neurexins participate in synapse maturation and maintenance. Alternative splicing of the neuroligin and neurexin genes results in multiple isoforms and presumably regulation of activity, while mutations appear to be associated with autism spectrum disorders. The crystal structures of the extracellular, cell adhesion domain of three neuroligins (NL1, NL2 and NL4) revealed features that distinguish the neuroligins from their enzyme relatives and could not be predicted by homology modelling from an AChE template. The structures of NL1 and NL4 bound with a soluble β-neurexin domain (Nrxβ1) revealed the precise position and orientation of the bound Nrxβ1 and the Ca2+-dependent interaction network at the complex interface. Herein we present an overview of the unbound and Nrxβ1-bound neuroligin structures and compare them with structures of AChEs with and without a bound fasciculin partner. This study exemplifies how an α/β-hydrolase fold domain tailored for catalysis varies to acquire adhesion properties, and defines three surface regions with distinctive locations and properties for homologous or heterologous partner association. [ABSTRACT FROM AUTHOR]

Summary: Background: Given the unusual nature of the schistosome surface (a highly unusual lipid bi-layer) and the central role of the schistosome tegument in host–parasite relations, an enhanced understanding of the lipid biochemistry of the schistosome surface can be expected to provide new insights into schistosome pathogenesis and lead to new interventions. Methods: Bioinformatics approaches including three-dimensional homology modeling, along with recombinant expression, dimensional gel electrophoresis, immunoblotting, and Southern hybridizations were employed to characterize a novel lysophospholipase gene transcript from Schistosoma japonicum. Results: A transcript encoding a small form lysophospholipase from the egg stage of S. japonicum was isolated as an expressed sequence tag (EST). The deduced polypeptide included 227 amino acid residues, shared identity with lysophospholipases of Schistosoma mansoni and Rattus norvegicus, and esterase A of Pseudomonas fluorescens, appeared to belong to the abhydrolase_2 family of phospholipases and carboxylesterases, and was structurally related to the α/β-hydrolases (pfam00561). The S. japonicum enzyme exhibited the GXSXG consensus active site characteristic of serine proteases, esterases, and lipases, and included the catalytic triad motif of Ser–Asp–His residues characteristic of serine hydrolases. Three-dimensional structural predictions accomplished using the coordinates of human acyl protein thioesterase and P. fluorescens esterase indicated that the putative catalytic triad formed by these three residues was located at the α/β-hydrolase fold characteristic of the lipases and esterases. Soluble S. japonicum lysophospholipase was expressed in Escherichia coli as a recombinant enzyme of ∼26kDa and employed to raise a mono-specific antiserum. Immunoblot analysis revealed a single 23-kDa band in both membrane-associated and soluble tissue fractions of adult schistosomes. Southern hybridization and bioinformatics analyses indicated the likely presence of allelic-specific polymorphisms and/or two copies of the lysophospholipase gene in the S. japonicum genome. Conclusions: A small form lysophospholipase has been characterized from the human schistosome, S. japonicum. The availability of the recombinant S. japonicum lysophospholipase should facilitate further characterization of the enzyme, including its substrate and inhibition profiles and its potential as an interventional target. Schistosome lysophospholipase may represent a new target for anti-schistosomal chemotherapy given that metrifonate, which targets the related enzyme acetylcholinesterase, is an effective and safe medicine for treatment of urinary schistosomiasis. [Copyright &y& Elsevier]

Abstract: Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase''s accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP. [Copyright &y& Elsevier]

1 H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) is a cofactor-devoid dioxygenase that is involved in the anthranilate pathway of quinaldine degradation. HOD has been proposed to belong to the α/β-hydrolase-fold superfamily of enzymes. N-terminally His6-tagged HOD has been crystallized by the hanging-drop vapour-diffusion method using sodium/potassium tartrate as a precipitant and CuCl2 as an additive. The structure was solved by the single anomalous dispersion (SAD) technique using data collected to 3.5 Å resolution at the Cu absorption peak wavelength. The crystals belong to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 153.788, c = 120.872 Å. [ABSTRACT FROM AUTHOR]

Abstract: J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase (GCA) isolated from Bacillus laterosporus J1, has been conventionally grouped as the only member of class V GCA, although its amino acid sequence shares less than 10% identity with members of other classes of GCA. Instead, it shows higher sequence similarities with Rhodococcus sp. strain MB1 cocaine esterase (RhCocE) and Acetobacter turbidans α-amino acid ester hydrolase (AtAEH), members of the α/β-hydrolase fold superfamily. Homology modeling and secondary structure prediction indicate that the N-terminal region of J1 acylase has an α/β-hydrolase folding pattern. The catalytic triads in RhCocE and AtAEH were identified in J1 acylase as S125, D264 and H309. Mutations to alanine at these positions were found to completely inactivate the enzyme. These results suggest that J1 acylase is a member of the α/β-hydrolase fold superfamily with a serine–histidine–aspartate catalytic triad. [Copyright &y& Elsevier]

Abstract: Metagenomic libraries give access to gene pool of bacteria present in environmental samples avoiding the culture bias. A metagenomic library of pond water microbial assemblage in plasmid vector containing about 532Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 11 unique lipolytic clones with an ability to hydrolyze tributyrin. DNA sequence of the lipolytic genes varied in G+C composition from 57% to 75%. Twelve lipolytic genes encoding proteins with 25–70% amino acid identity with proteins in the databases were identified. Ten of the encoded proteins belonged to seven known lipolytic protein families. One of the proteins was similar to recently identified esterase BioH. A lipolytic protein with high similarity to yet uncharacterized α/β hydrolase protein family abh_upf0017 was identified from one of the clones. Conserved motif for lipolytic enzymes GXSXG, conserved aspartic and histidine residues were identified in this encoded protein. [Copyright &y& Elsevier]

Presents a study which sought to comprehensively determine the kinetic parameters and complex structures of Pseudomonas fluorescens IP01 (CumD) with various substrates and organic acids, in order to determine the mechanism underlying the substrate specificity of meta-cleavage product-hydrolases. Kinetic parameters of CumD for various substrates; Complex structures of CumD with various organic acids; Binding direction of benzoate.

Our understanding of the genes involved in Alzheimer''s disease (AD) is incomplete. Using subtractive cloning technology, we discovered that the α/β-hydrolase fold protein gene NDRG2 (NDRG family member 2) is upregulated at both the RNA and protein levels in AD brains. Expression of NDRG2 in affected brains was revealed in (1) cortical pyramidal neurons, (2) senile plaques and (3) cellular processes of dystrophic neurons. Overexpression of two splice variants encoding a long and short NDRG2 isoform in hippocampal pyramidal neurons of transgenic mice resulted in localization of both isoforms to dendritic processes. Taken together, our findings suggest that NDRG2 upregulation is associated with disease pathogenesis in the human brain and provide new insight into the molecular changes that occur in AD. [Copyright &y& Elsevier]

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase (CumD) from Pseudomonas fluorescens IP01 hydrolyzes a meta-cleavage product generated in the cumene (isopropylbenzene) degradation pathway. The crystal structures of the inactive S103A mutant of the CumD enzyme complexed with isobutyrate and acetate ions were determined at 1.6 and 2.0 Å resolution, respectively. The isobutyrate and acetate ions were located at the same position in the active site, and occupied the site for a part of the hydrolysis product with CumD, which has the key determinant group for the substrate specificity of related hydrolases. One of the oxygen atoms of the carboxyl group of the isobutyrate ion was hydrogen bonded with a water molecule and His252. Another oxygen atom of the carboxyl group was situated in an oxyanion hole formed by the two main-chain N atoms. The isopropyl group of the isobutyric acid was recognized by the side-chains of the hydrophobic residues. The substrate-binding pocket of CumD was long, and the inhibition constants of various organic acids corresponded well to it. In comparison with the structure of BphD from Rhodococcus sp. RHA1, the structural basis for the substrate specificity of related hydrolases, is revealed. [ABSTRACT FROM AUTHOR]