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Actinobacteria cells Rhodococcus erythropolis 4-1 and Rhodococcus erythropolis 11-2 and Proteobacteria Alcaligenes faecalis 2, which have amidase activity, were immobilized by entrapping barium alginate and agarose into the gel structure, as well as by obtaining biofilms on thermally expanded graphite (TEG). The operational stability of such immobilized biocatalysts after storage in frozen and dehydrated form was determined, and a prototype of a conductometric acrylamide biosensor based on such a bioselective agent was developed. The most preferred method for storing immobilized cells was freezing at temperatures from –20 to –80°C; long-term storage is also possible wet at 4–25°C. It was shown that these cells were most preferable for the biodetection of acrylamide A. faecalis 2, immobilized in an agarose gel structure. An agarose gel with bacterial cells immobilized in its structure had greater mechanical strength and stability during successive cycles of conversion of acrylamide into acrylic acid compared to barium alginate gel. The mechanical strength of barium alginate gel can be enhanced by the addition of carbon nanomaterials during cell immobilization. Growing biofilms on carbon materials used for manufacturing electrodes is also promising. Biofilms of R. erythropolis 11-2 on TEG are capable of converting acrylamide into acrylic acid in more than 20 reaction cycles while maintaining at least 50% amidase activity. [ABSTRACT FROM AUTHOR]

While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG‐SP‐1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site‐saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3‐ and 8‐fold improved activity against highly stable N‐aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester‐PU and a PA (nylon 6) by the activity of a single, metagenome‐derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG‐SP‐1 beyond the reported low‐molecular weight carbamates. Together, these findings promise advanced strategies for the bio‐based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers. [ABSTRACT FROM AUTHOR]

Während Kunststoffe wie Polyethylenterephthalat (PET) bereits effizient durch die Aktivität von Hydrolasen abgebaut werden können, sind andere synthetische Polymere wie Polyurethane (PUs) und Polyamide (PAs) weitgehend resistent gegenüber einem biologischen Abbau. In dieser Studie lösten wir die erste Kristallstruktur der metagenomischen Urethanase UMG‐SP‐1, identifizierten hochflexible Loopregionen, die Reste des aktiven Zentrums enthalten, und untersuchten insgesamt 20 potenzielle Hotspots mittels Sättigungsmutagenese. Die durch Protein Engineering erzeugten Einzelmutanten wiesen eine fast 3‐ bzw. 8‐fach verbesserte Aktivität gegenüber hochstabilen N‐Arylurethan‐ und Amidbindungen auf. Darüber hinaus konnte die Freisetzung der entsprechenden Monomere aus einem thermoplastischen Polyester‐PU und einem PA (Nylon 6) durch die Aktivität einer einzigen, metagenomischen Urethanase nach kurzer Inkubationszeit nachgewiesen werden. Dadurch konnte das Hydrolyseprofil von UMG‐SP‐1 über die bekannten niedermolekularen Carbamate hinaus erweitert werden und erschließt so neue Möglichkeiten für den enzymatischen Abbau und das Recycling von Kunststoffen und Plastikabfällen. Damit unterstützt diese Studie Bemühungen um die Verbesserung einer Kreislaufwirtschaft für synthetische Polymere. [ABSTRACT FROM AUTHOR]

Hydroxamic acids with functional group R-CONHOH, have been used as possible inhibitors of metalloenzymes because of their innate ability to strongly bind with metal ions. The synthesis from amides to hydroxamic acids is commonly carried out via a synthetic route wherein hazardous chemicals like acids and extreme reaction conditions are used. Hence, the present study aims to develop a biocatalytic process for the synthesis of a series of hydroxamic acids using the resting cells of Bacillus smithii IIIMB2907. Further, through in silico analysis of the synthesized compounds using molecular docking, molecular dynamics pharmacophore analysis, and density functional theory (DFT) calculations, it was observed that ligand N 1-(3-(trifluoromethyl)phenyl)- N 8-hydroxyoctanediamide (2c) showed better interaction with class I histone deacetylases (HDACs) on comparisons with FDA approved anti-cancer drug Suberoylanilide hydroxamic acid (SAHA). Furthermore, in vitro HDAC activity of 2c in C2C12 skeletal muscle cell lines validates the potency of this molecule as an HDAC inhibitor, as it shows 33 % HDAC inhibition on comparison with SAHA which showed only 14 % HDAC inhibition. Therefore, this study proposes a novel process for the synthesis of a potential drug candidate that may act as an inhibitor of class I HDAC's. [Display omitted] • Development of a biocatalytic process for the synthesis of hydroxamic acids. • In silico analysis of a potent compound with class I Histone Deacetylases (HDAC). • 2.4 fold increased HDAC inhibition (in vitro) of the potent compound on comparison with SAHA. [ABSTRACT FROM AUTHOR]

To isolate the iprodione-degrading microbial resources suitable for Qinghai-Tibet Plateau, three efficient iprodione-degrading strain Y-20, Y-29 and Y-32 were isolated from the greenhouse soil in Tibet by enrichment culture method. These strains were preliminarily identified based on the morphological characteristics, physiological and biochemical characteristics, and 16S rRNA gene sequence analysis. The effects of NaCl concentration（m/V）, pH value, temperature, inoculation amount（V/V）and initial iprodione concentration on the iprodione)degradation rates of these strains were also investigated. The metabolic pathways of iprodione and ipaH gene in these strains were analyzed using gas chromatography method and polymerase chain reaction technique, respectively. The results showed that strain Y-20, Y-29 and Y-32 were identified as three species of Microbacterium genus. Under the optimal iprodione-degrading condition of 1.0% NaCl（m/V）, pH 7.0, 25-30℃, 5% inoculation amount（V/V）and 100 mg/L initial iprodione concentration, 100 mg/L iprodione was completely degraded by these strains within 8-12 h. These strains decomposed iprodione into N-（3,5-dichlorophenyl）-2,4-dioximidazolidine and isopropyl carbamate, and had a highly similar ipaH gene（99.14%-99.69%）. This study provides the strain resources and theoretical basis for the bioremediation of iprodione-contaminated environment at high altitude area. [ABSTRACT FROM AUTHOR]

Geobacillus thermoleovorans MTCC 13131, an amide hydrolyzing bacteria was isolated from a hot spring in Himachal Pradesh and identified through 16S rRNA gene sequence analysis. The amidase derived from this bacterium exhibited hydrolyzing catalytic ability against aliphatic and aromatic amides. The isolate was characterized for morphological and biochemical properties. Further, the production of amidase enzyme from this isolate was evaluated using approach of one-variable-at-a-time and response surface method. The Response Surface Methodology based study indicated the importance of nitrogen sources and growth period for amidase production. Optimal production was achieved at a temperature 55 °C, and production pH 7.5 in the production medium comprising diammonium hydrogen phosphate (0.4%), peptone (0.45%) and yeast extract (0.3%). The wide substrate affinity of the strain suggests its potential role in biotransformation of amides to corresponding acids of industrial significance along with its strong capacity to degrade the toxic amide in polluted environmental samples. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae (Group B Streptococcus , GBS) is primarily known as a major neonatal pathogen. In adults, these bacteria often colonize the gastrointestinal and urogenital tracts. Treatment of infections using antibiotics is often complicated by recurrences caused by multi-resistant streptococci. Endolysin EN534 from prophage A2 of human isolate Streptococcus agalactiae KMB-534 has a modular structure consisting of two terminal catalytic domains, amidase_3 and CHAP, and one central binding domain, LysM. The EN534 gene was cloned into an expression vector, and the corresponding recombinant protein EN534-C was expressed in Escherichia coli in a soluble form and isolated by affinity chromatography. The lytic activity of this endolysin was tested on cell wall substrates from different GBS serotypes, B. subtilis , L. jensenii, and E. coli. The enzyme lysed streptococci, but not beneficial vaginal lactobacilli. The isolated protein is stable in a temperature range of 20–37 °C. Calcium ions enhanced the activity of the enzyme in the pH range from 5.0 to 8.0. The exolytic activity of EN534-C was observed by time-lapse fluorescence microscopy on a S. agalactiae CCM 6187 substrate. Recombinant endolysin EN534-C may have the potential to become an antimicrobial agent for the treatment of S. agalactiae infections. [Display omitted] • Endolysin EN534-C from streptococcal prophage was cloned and characterized. • EN534-C contains three domains - catalytic amidase_3 and CHAP and binding LysM. • EN534-C showed significant exolytic activity towards human pathogen S. agalactiae GBS. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum is one of the most notorious and ubiquitous soilborne plant pathogens, causing serious economic losses to a large number of hosts worldwide. Although virulence factors have been identified in this filamentous fungus, including various cell-wall-degrading enzymes, toxins, oxalic acids and effectors, our understanding of its virulence strategies is far from complete. To explore novel factors contributing to disease, a new pipeline combining forward genetic screening and next-generation sequencing was utilized in this study. Analysis of a hypovirulent mutant revealed that a mutation in an amidase-encoding gene, Sscle_10g079050, resulted in reduced virulence. This is a first report on the contribution of an amidase to fungal virulence, likely through affecting oxalic acid homeostasis. [ABSTRACT FROM AUTHOR]

Construction and remodeling of the bacterial peptidoglycan (PG) cell wall must be carefully coordinated with cell growth and division. Central to cell wall construction are hydrolases that cleave bonds in peptidoglycan. These enzymes also represent potential new antibiotic targets. One such hydrolase, the amidase LytH in Staphylococcus aureus, acts to remove stem peptides from PG, controlling where substrates are available for insertion of new PG strands and consequently regulating cell size. When it is absent, cells grow excessively large and have division defects. For activity, LytH requires a protein partner, ActH, that consists of an intracellular domain, a large rhomboid protease domain, and three extracellular tetratricopeptide repeats (TPRs). Here, we demonstrate that the amidase-activating function of ActH is entirely contained in its extracellular TPRs. We show that ActH binding stabilizes metals in the LytH active site and that LytH metal binding in turn is needed for stable complexation with ActH. We further present a structure of a complex of the extracellular domains of LytH and ActH. Our findings suggest that metal cofactor stabilization is a general strategy used by amidase activators and that ActH houses multiple functions within a single protein. [ABSTRACT FROM AUTHOR]

Urethanase (EC 3.5.1.75) can reduce ethyl carbamate (EC), a group 2A carcinogen found in foods and liquor. However, it is not yet commercially available. Urethanase has been detected as an intracellular enzyme from yeast, filamentous fungi, and bacteria. Based on the most recent progress in the sequence analysis of this enzyme, it was observed that amidase-type enzyme can degrade EC. All five enzymes had highly conserved sequences of amidase signature family, and their molecular masses were in the range of 52–62 kDa. The enzymes of Candida parapsilosis and Aspergillus oryzae formed a homotetramer, and that of Rhodococcus equi strain TB-60 existed as a monomer. Most urethanases exhibited amidase activity, and those of C. parapsilosis and A. oryzae also demonstrated high activity against acrylamide, which is a group 2A carcinogen. It was recently reported that urease and esterase also exhibited urethanase activity. Although research on the enzymatic degradation of EC has been very limited, recently some sequences of EC-degrading enzyme have been elucidated, and it is anticipated that new enzymes would be developed and applied into practical use. Key points: • Recently, some urethanase sequences have been elucidated • The amino acid residues that formed the catalytic triad were conserved • Urethanase shows amidase activity and can also degrade acrylamide [ABSTRACT FROM AUTHOR]

Metopimazine (MPZ) is a peripherally restricted, dopamine D2 receptor antagonist used for four decades to treat acute nausea and vomiting. MPZ is currently under clinical investigation for the treatment of gastroparesis (GP). MPZ undergoes high first‐pass metabolism that produces metopimazine acid (MPZA), the major circulating metabolite in humans. Despite a long history of use, the enzymes involved in the metabolism of MPZ have not been identified. Here we report a series of studies designed to identify potential MPZ metabolites in vitro, determine their clinical relevance in humans, and elucidate the enzymes responsible for their formation. The findings demonstrated that the formation of MPZA was primarily catalyzed by human liver microsomal amidase. Additionally, human liver cytosolic aldehyde oxidase (AO) catalyzes the formation of MPZA, in vitro, although to a much lesser extent. Neither cytochrome P450 enzymes nor flavin‐monooxygenases (FMO) were involved in the formation MPZA, although two minor oxidative pathways were catalyzed by CYP3A4 and CYP2D6 in vitro. Analysis of plasma samples from subjects dosed 60 mg of MPZ verified that these oxidative pathways are very minor and that CYP enzyme involvement was negligible compared to microsomal amidase/hydrolase in overall MPZ metabolism in humans. The metabolism by liver amidase, an enzyme family not well defined in small molecule drug metabolism, with minimal metabolism by CYPs, differentiates this drug from current D2 antagonists used or in development for the treatment of GP. [ABSTRACT FROM AUTHOR]

Metopimazine (MPZ) is a peripherally restricted, dopamine D2 receptor antagonist used for four decades to treat acute nausea and vomiting. MPZ is currently under clinical investigation for the treatment of gastroparesis (GP). MPZ undergoes high first-pass metabolism that produces metopimazine acid (MPZA), the major circulating metabolite in humans. Despite a long history of use, the enzymes involved in the metabolism of MPZ have not been identified. Here we report a series of studies designed to identify potential MPZ metabolites in vitro, determine their clinical relevance in humans, and elucidate the enzymes responsible for their formation. The findings demonstrated that the formation of MPZA was primarily catalyzed by human liver microsomal amidase. Additionally, human liver cytosolic aldehyde oxidase (AO) catalyzes the formation of MPZA, in vitro, although to a much lesser extent. Neither cytochrome P450 enzymes nor flavin-monooxygenases (FMO) were involved in the formation MPZA, although two minor oxidative pathways were catalyzed by CYP3A4 and CYP2D6 in vitro. Analysis of plasma samples from subjects dosed 60 mg of MPZ verified that these oxidative pathways are very minor and that CYP enzyme involvement was negligible compared to microsomal amidase/hydrolase in overall MPZ metabolism in humans. The metabolism by liver amidase, an enzyme family not well defined in small molecule drug metabolism, with minimal metabolism by CYPs, differentiates this drug from current D2 antagonists used or in development for the treatment of GP. [ABSTRACT FROM AUTHOR]

Peptidoglycan (PG), a heteropolysaccharide component of the mycobacterial cell wall can be shed during tuberculosis infection with immunomodulatory consequences. As such, changes in PG structure are expected to have important implications on disease progression and host responses during infection with Mycobacterium tuberculosis. Mycobacterial amidases have important roles in remodeling of PG during cell division and are implicated in susceptibility to antibiotics. However, their role in modulating host immunity remains unknown. We assessed the bacterial burden and host immune responses to M. tuberculosis mutants defective for either one of two PG N-acetylmuramyl-L-alanine amidases, Ami1 and Ami4, in bone marrow-derived macrophages (BMDM) and C57BL/6 mice. In infected BMDM, the single deletion of both genes resulted in increased proinflammatory cytokine responses. In mice, infection with the Δami1 mutant led to differential induction of pro-inflammatory cytokines and chemokines, decreased cellular recruitment and reduced lung pathology during the acute phase of the infection. While increased proinflammatory cytokines production was observed in BMDM infected with the Δami4 mutant, these effects did not prevail in mice. Infection using the Δami1 and Δami4 Mtb mutants showed that these genes are dispensable for intracellular mycobacterial growth in macrophages and mycobacterial burden in mice. These findings suggest that both Ami1 and Ami4 in M. tuberculosis are not essential for mycobacterial growth within the host. In summary, we show that amidases are important for modulating host immunity during Mtb infection in murine macrophages and mice. [ABSTRACT FROM AUTHOR]

Phage-derived endolysins, enzymes that degrade peptidoglycans, have the potential to serve as alternative antimicrobial agents. Psa, which was identified as an endolysin encoded in the genome of Clostridium perfringens st13, was shown to specifically lyse C. perfringens. Psa has an N-terminal catalytic domain that is homologous to the Amidase_2 domain (PF01510), and a novel C-terminal cell wall-binding domain. Here, we determined the X-ray structure of the Psa catalytic domain (Psa-CD) at 1.65 Å resolution. Psa-CD has a typical Amidase_2 domain structure, consisting of a spherical structure with a central β-sheet surrounded by two α-helix groups. Furthermore, there is a Zn2+ at the center of Psa-CD catalytic reaction site, as well as a unique T-shaped substrate-binding groove consisting of two grooves on the molecule surface. We performed modeling study of the enzyme/substrate complex along with a mutational analysis, and demonstrated that the structure of the substrate-binding groove is closely related to the amidase activity. Furthermore, we proposed a Zn2+-mediated catalytic reaction mechanism for the Amidase_2 family, in which tyrosine constitutes part of the catalytic reaction site. [Display omitted] • Psa is a Clostridium perfringens -specific endolysin with an Amidase_2 domain. • The catalytic domain structure of Psa was determined by X-ray structure analysis. • Enzyme/substrate complex modeling and mutational analysis were performed. • The structure of the substrate-binding groove is closely related to lytic activity. • The catalytic reaction mechanism for the Amidase_2 family was proposed. [ABSTRACT FROM AUTHOR]

Both enantiomers of cis‐1,2‐disubstituted cyclohexane have been obtained enantioselectively through engineered amidase‐catalyzed desymmetrization of meso carbocyclic 1,2‐dicarboxamides under mild condition. Based on the enzyme‐substrate binding model suggested by molecular docking, the amplification and reversal of enantioselectivity of amidase was quickly achieved through generating and testing only 8 variants. The origin of enantiopreference for amidase was revealed by MD simulations and the reason for the reversal of enantioselectivity towards amidase variant was disclosed. The synthetic application of biocatalytic meso‐desymmetrization was demonstrated by straightforward transformation of the products to enantiomerically pure heterocyclic compounds. [ABSTRACT FROM AUTHOR]

Ethyl carbamate (EC), widely found in alcoholic beverages, has been revealed to be a probable carcinogen in humans. Urethanase (EC 3.5.1.75) is an effective enzyme for the degradation of EC; however, the previously identified urethanases exhibited insufficient acid and alcohol resistance. In this study, an enantioselective amidase (AmdA) screened from Agrobacterium tumefaciens d 3 exhibited urethanase activity with excellent alcohol resistance. AmdA was first overexpressed in Escherichia coli ; however, the recombinant protein was primarily located in inclusion bodies, and thus, co-expression of molecular chaperones was used. The activity of AmdA increased 3.1 fold to 307 U/L, and the specific activity of urethanase with C-terminal His-tags reached 0.62 U/mg after purification through a Ni–NTA column. Subsequently, the enzymatic properties and kinetic constants of AmdA were investigated. The optimum temperature for AmdA was 55 °C, it showed the highest activity at pH 7.5, and the K m was 0.964 mM. Moreover, after 1 h of heat treatment at 37 °C in a 5–20% (v/v) ethanol solution, the residual urethanase activity was higher than 91%, considerably more than that reported thus far. [ABSTRACT FROM AUTHOR]

N-Acetylmuramoyl-L-alanine amidases are periplasmic hydrolases that cleave the amide bond between N-acetylmuramic acid and alanine in peptidoglycan (PG). Unlike many Gram-negative bacteria that encode redundant periplasmic amidases, Vibrio fischeri appears to encode a single protein that is homologous to AmiB of Vibrio cholerae. We screened a V. fischeri transposon mutant library for strains altered in biofilm production and discovered a biofilm-overproducing strain with an insertion in amiB (VF_2326). Further characterization of biofilm enhancement suggested that this phenotype was due to the overproduction of cellulose, and it was dependent on the bcsA cellulose synthase. Additionally, the amiB mutant was nonmotile, perhaps due to defects in its ability to septate during division. The amidase mutant was unable to compete with the wild type for the colonization of V. fischeri's symbiotic host, the squid Euprymna scolopes. In single-strain inoculations, host squid inoculated with the mutant eventually became colonized but with a much lower efficiency than in squid inoculated with the wild type. This observation was consistent with the pleiotropic effects of the amiB mutation and led us to speculate that motile suppressors of the amiB mutant were responsible for the partially restored colonization. In culture, motile suppressor mutants carried point mutations in a single gene (VF_1477), resulting in a partial restoration of wild-type motility. In addition, these point mutations reversed the effect of the amiB mutation on cellulosic biofilm production. These data are consistent with V. fischeri AmiB possessing amidase activity; they also suggest that AmiB suppresses cellulosic biofilm formation but promotes successful host colonization. [ABSTRACT FROM AUTHOR]

Peptidoglycan (PG), a heteropolysaccharide component of the mycobacterial cell wall can be shed during tuberculosis infection with immunomodulatory consequences. As such, changes in PG structure are expected to have important implications on disease progression and host responses during infection with Mycobacterium tuberculosis. Mycobacterial amidases have important roles in remodeling of PG during cell division and are implicated in susceptibility to antibiotics. However, their role in modulating host immunity remains unknown. We assessed the bacterial burden and host immune responses to M. tuberculosis mutants defective for either one of two PG N-acetylmuramyl-L-alanine amidases, Ami1 and Ami4, in bone marrow-derived macrophages (BMDM) and C57BL/6 mice. In infected BMDM, the single deletion of both genes resulted in increased proinflammatory cytokine responses. In mice, infection with the Δami1 mutant led to differential induction of pro-inflammatory cytokines and chemokines, decreased cellular recruitment and reduced lung pathology during the acute phase of the infection. While increased proinflammatory cytokines production was observed in BMDM infected with the Δami4 mutant, these effects did not prevail in mice. Infection using the Δami1 and Δami4 Mtb mutants showed that these genes are dispensable for intracellular mycobacterial growth in macrophages and mycobacterial burden in mice. These findings suggest that both Ami1 and Ami4 in M. tuberculosis are not essential for mycobacterial growth within the host. In summary, we show that amidases are important for modulating host immunity during Mtb infection in murine macrophages and mice. [ABSTRACT FROM AUTHOR]

Aim: Presently, N‐hydroxy‐N′‐phenyloctanediamide (vorinostat) which is an effective histone deacetylase inhibitor, is being synthesized chemically. Hence, present study aims to develop an eco‐friendly approach for the synthesis of vorinostat from N′‐phenyloctanediamide through biotransformation. Methods and Results: Using the amidase of Bacillus smithii IIIMB2907 in time course conversion and organic solvent compatibility, maximum bioconversion was observed at 12 h of reaction time and in presence of ethanol, respectively. Potassium phosphate buffer of pH 7·0 supported maximum bioconversion of N′‐phenyloctanediamide (10 mmol l−1) into N‐hydroxy‐N′‐ phenyloctanediamide at 40°C. Bench scale study was successfully carried out with 83% yield of purified vorinostat. Conclusion: In this study, an eco‐friendly approach for the biotransformation of N′‐phenyloctanediamide into vorinostat was developed by using cell free extract of thermophilic strain B. smithii IIIMB2907. Significance and Impact of the Study: Microbial amidase has achieved remarkable attention in the field of biotransformation for the green synthesis of hydroxamic acids. Utilization of amidase from B. smithii IIIMB2907, specifically in the synthesis of vorinostat drug is a foremost attempt in the development a novel process and can also be employed in the synthesis of its derivatives as well. [ABSTRACT FROM AUTHOR]

Urethanase (EC 3.5.1.75) catalyzes the hydrolysis of ethyl carbamate (EC) to ethanol, carbon dioxide, and ammonia. From our recent study, we expected that an acetamidase encoded by amdS of Aspergillus oryzae may catalyze the degradation of EC because it is homologous with a Candida parapsilosis urethanase (CPUTNase) recently identified. Urethanase is a prospective candidate to reduce EC in alcoholic beverages, but knowledge of this enzyme is very limited. Recombinant AmdS was expressed to study its enzymatic properties. Purified AmdS was identified as a homo-tetramer consisting of four 60 kDa units and exhibited urethanase activity. In a 20% ethanol solution, AmdS had 65% activity compared with a solution without ethanol. Residual activity after 18 h indicated that AmdS was stable in 0%–40% ethanol solutions. The optimum temperature of AmdS was 40 °C. This enzyme showed urethanase activity at pH 6.4–9.6 and exhibited its highest activity at pH 9.6. The K m value of AmdS for EC was 8.2 mM, similar to the K m value (7.6 mM) of CPUTNase. AmdS showed activity not only for EC and acetamide but also other amide compounds. In this study, we investigated the enzymatic properties of AmdS that was identified as acetamidase and showed that an amidase can be an enzymatic candidate that degrades EC. [ABSTRACT FROM AUTHOR]

Peptidoglycan recognition proteins (PGRPs), which are structurally conserved innate immune molecules in invertebrate and vertebrate animals, play the important roles in regulation of innate immune responses. In this paper, three PGRP genes of spotted sea bass, Lateolabrax maculatus , were cloned, designated as Ssb-PGRP2 , Ssb - PGRP-L2 and Ssb-PGRP-SC2 , respectively. Sequence analysis showed that the deduced amino acid sequences of Ssb-PGRP2, Ssb-PGRP-L2 and Ssb-PGRP-SC2 proteins contained respectively 468, 482 and 167 amino acid residues, and had the typical structural features of PGRPs, i.e. conserved PGRP domain and Zn2+ binding domain including four specific amino acid residues which were required for amidase activity. q-PCR analysis of total mRNA showed that the mRNA expression of three PGRP genes were detected in all the examined tissues and the expression patterns of Ssb-PGRP2 , Ssb-PGRP-L2 and Ssb-PGRP-SC2 were different. After injected with LPS, Poly (I:C) and Edwardsiella tarda , there was a clear time-dependent expression pattern for each of the three PGRP genes in head kidney, spleen, intestine and gill of the spotted sea bass. In our study, three recombinant proteins corresponding to the three members of the peptidoglycan recognition protein family were expressed and purified. Moreover, all of the three recombinant PGRP proteins significantly inhibited bacterial survival and growth, and expressed bactericidal effects on Vibrio harveyi , Staphylococcus aureus and Edwardsiella tarda. In particular, it was firstly verified that their antimicrobial activity presented the superimposed effect. Overall, these findings indicated that three PGRP genes of spotted sea bass were at least involved in host defense against bacterial infections. • Three members of the PGRP family were identified in spotted sea bass. • The three members were up-regulated by LPS, Poly (I:C) and Edwardsiella tarda. • All of the three recombinant PGRP proteins possessed the amidase activity. • All of the three recombinant proteins expressed bactericidal effects. • Their antimicrobial activity presented the superimposed effect. [ABSTRACT FROM AUTHOR]

Urethanase (EC 3.5.1.75) is an effective enzyme for removing ethyl carbamate (EC) present in alcoholic beverages. However, urethanase is not well studied and has not yet been developed for practical use. In this study, we report a new urethanase (CPUTNase) from the yeast Candida parapsilosis. Because C. parapsilosis can assimilate EC as its sole nitrogen source, the enzyme was extracted from yeast cells and purified using ion-exchange chromatography. The CPUTNase was estimated as a homotetramer comprising four units of a 61.7 kDa protein. In a 20% ethanol solution, CPUTNase had 73% activity compared with a solution without ethanol. Residual activity after 18 h indicated that CPUTNase was stable in 0%–40% ethanol solutions. The optimum temperature of CPUTNase was 43°C. This enzyme showed urethanase activity at pH 5.5–10.0 and exhibited its highest activity at pH 10. The gene of CPUTNase was identified, and a recombinant enzyme was expressed in the yeast Saccharomyces cerevisiae. Characteristics of recombinant CPUTNase were identical to the native enzyme. The putative amino acid sequence indicated that CPUTNase was an amidase family protein. Further, substrate specificity supported this sequence analysis because CPUTNase showed higher activities toward amide compounds. These results suggest that amidase could be a candidate for urethanase. We discovered a new enzyme and investigated its enzymatic characteristics, sequence, and recombinant CPUTNase expression. These results contribute to a further understanding of urethanase. [ABSTRACT FROM AUTHOR]

Amidase is an important hydrolytic enzyme in detoxification metabolism. Amidase hydrolyzes a wide variety of nonpeptide carbon‑nitrogen bonds by attacking a cyano group or carbonyl carbon. However, little is known about the relationship between amidase and insecticides. In this study, the amidase activity was significantly higher in cyflumetofen-resistant strain (CyR) than in the susceptible strain (SS) of Tetranychus cinnabarinus , and diethyl-phosphoramidate (an amidase inhibitor) significantly decreased cyflumetofen resistance in T. cinnabarinus. More importantly, an amidase gene, TcAmidase01 , was identified in T. cinnabarinus , and the TcAmidase01 overexpression was detected in both two cyflumetofen-resistant strains (CyR and YN-CyR), indicating that it is involved in cyflumetofen resistance in mites. A phylogenetic analysis showed that TcAmidase01 was clustered with deaminated glutathione amidases, which possess hydrolytic activity. The recombinant TcAmidase01 protein showed amidase activity toward succinamate, and the activity could be inhibited by cyflumetofen. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis provided evidence that recombinant TcAmidase01 could decompose cyflumetofen by hydrolysis, and the potential metabolites (2-(4-(tert-butyl) phenyl)-2-cyanoacetate and 2-(trifluoromethyl) benzoic acid) were identified. These results show that TcAmidase01 contribute to cyflumetofen-resistance in T. cinnabarinus by hydrolyzing cyflumetofen, and this is the first study to suggest that amidase has a role in insecticides resistance in arthropods. Unlabelled Image • The bioassay suggested that amidases contribute to cyflumetofen resistance. • The mRNA level of TcAmidase01 was overexpression in cyflumetofen resistant strains. • Recombinant TcAmidase01 hydrolyzed cyflumetofen and metabolites were identified. [ABSTRACT FROM AUTHOR]

A highly active secretory expression system in Bacillus subtilis was constructed for enhancing the heterogeneous secretory expression efficiency of a recombinant amidase from Bacillus megaterium (Bm -Ami). Initially, six promoters P lytR , P spoVG , P aprE , P yvyD , P ftsH , P amyE were screened out from 33 endogenous phase-dependent promoters via the strategy of promoter engineering which showed higher transcriptional levels than the original promoter P cdd. Afterwards, a secretory expression system pBSHdd2-20 containing a dual-promoter P amyE-cdd and a signal peptide Pac was constructed showing an extracellular Bm -Ami activity of 135.58 U mL−1 during shake-flask cultivation, which was 3.58-fold greater than that with plasmid pBSH1 (control). Finally, extracellular amidase activity of a strain containing the plasmid pBSHdd2-20 reached 430 U mL−1 (equal to 10.8 mg mL−1) and 10.72 U mg−1 DCW after 52 h in a scale-up fermentation, which was the highest secretory efficiency for Bm -Ami in all studies to date. In conclusion, a highly secretory system constructed in this study can be applied for the industrial production of amidase and provides a basis for the development of an excellent secretory expression system for similar proteins. [ABSTRACT FROM AUTHOR]

Amidase from Bacillus sp. APB-6 with very good acyltransferase activity was purified to homogeneity with a purification fold of 3.68 and 53.20% enzyme yield. The purified protein's subunit molecular mass was determined approximately 42 kDa. Hyperactivity of the enzyme was observed at pH 7.5 (150 mM, potassium-phosphate buffer) and 50 °C of incubation. An enhancement in activity up to 42% was recorded with ethylenediaminetetraacetic acid and dithiothreitol. The kinetic parameter Km values for substrates: acetamide and hydroxylamine-hydrochloride were 73.0 and 153 mM, respectively. Further, the Vmax for acyltransferase activity was 1667 U/mg of protein and the Ki for acetamide was calculated as 37.0 mM. The enzyme showed tolerance to various organic solvents (10%, v/v) and worked well in the biphasic reaction medium. The acyltransferase activity in presence of solvents i.e. biphasic medium may prove highly favorable for the transformation of hydrophobic amides, which otherwise is not possible in simple aqueous phase. [ABSTRACT FROM AUTHOR]

During bacterial cell division, hydrolysis of septal peptidoglycan (sPG) is crucial for cell separation. This sPG hydrolysis is performed by the enzyme amidases whose activity is regulated by the integral membrane protein complex FtsEX-EnvC. FtsEX is an ATP-binding cassette transporter, and EnvC is a long coiled-coil protein that interacts with and activates the amidases. The molecular mechanism by which the FtsEX-EnvC complex activates amidases remains largely unclear. We present the cryo-electron microscopy structure of the FtsEX-EnvC complex from the pathogenic bacteria V. cholerae (FtsEX-EnvC VC). FtsEX-EnvC VC in the presence of ADP adopts a distinct conformation where EnvC is "horizontally extended" rather than "vertically extended". Subsequent structural studies suggest that EnvC can swing between these conformations in space in a nucleotide-dependent manner. Our structural analysis and functional studies suggest that FtsEX-EnvC VC employs spatial control of EnvC for amidase activation, providing mechanistic insights into the FtsEX-EnvC regulation on septal peptidoglycan hydrolysis. [Display omitted] • Cryo-EM structure of FtsEX-EnvC from V. cholerae with ADP bound • EnvC is oriented nearly parallel to the membrane, revealing a novel conformation • EnvC swings up and down in space in a nucleotide-dependent manner • FtsEX can control EnvC through spatial separation to regulate peptidoglycan hydrolysis The FtsEX-EnvC complex plays a crucial role in bacterial cell division by regulating septal peptidoglycan hydrolysis. Hao et al. determined the cryo-EM structure of the FtsEX-EnvC complex from Vibrio cholerae with ADP bound, offering mechanistic insights into how the complex controls septal peptidoglycan hydrolysis. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) is one of the common causes of implant associated biofilm infections and their biofilms are resistant to antibiotics. S. aureus amidase (AM) protein, a cell wall hydrolase that cleaves the amide bond between N -acetylmuramic acid and L-alanine residue of the stem peptide, is several fold over-expressed under biofilm conditions. Previous studies demonstrated an autolysin mutant in S. aureus that lacks the AM protein, is highly impaired in biofilm development. We carried out a structure-based small molecule design using the crystal structure of AM protein catalytic domain to identify inhibitors that can block amidase activity and therefore inhibits S. aureus biofilm formation. Sequential virtual screening followed by pharmacokinetic analysis and bioassay studies filtered 25 small molecules from different databases. Two compounds from the SPECS database, SPECS-1 and SPECS-2, were selected based on the best docking score and minimum biofilm inhibitory concentration towards S. aureus biofilms. SPECS-1 and SPECS-2 were further tested for their structural/energetic stability in complex with the AM protein using molecular dynamics simulation and MM-GBSA techniques. In vitro, biofilm inhibition studies on different surfaces confirmed that treatment with SPECS-1 and SPECS-2 at a concentration of 250 μg/ml exhibited significant prevention and disruption of S. aureus biofilms. Finally, the in vitro anti-biofilm activities of these two compounds were validated against Methicillin-resistant S. aureus clinical isolates. We concluded that the discovered compounds SPECS-1 and SPECS-2 are safe and exhibit biofilm preventive and disruption activity for inhibiting the S. aureus biofilms and hence can be used to treat implant-associated biofilm infections. [ABSTRACT FROM AUTHOR]

The crystallization of amidase, the ultimate enzyme in the Trp‐dependent auxin‐biosynthesis pathway, from Arabidopsis thaliana was attempted using protein samples with at least 95% purity. Cube‐shaped crystals that were assumed to be amidase crystals that belonged to space group I4 (unit‐cell parameters a = b = 128.6, c = 249.7 Å) were obtained and diffracted to 3.0 Å resolution. Molecular replacement using structures from the PDB containing the amidase signature fold as search models was unsuccessful in yielding a convincing solution. Using the Sequence‐Independent Molecular replacement Based on Available Databases (SIMBAD) program, it was discovered that the structure corresponded to dihydrolipoamide succinyltransferase from Escherichia coli (PDB entry 1c4t), which is considered to be a common crystallization contaminant protein. The structure was refined to an Rwork of 23.0% and an Rfree of 27.2% at 3.0 Å resolution. The structure was compared with others of the same protein deposited in the PDB. This is the first report of the structure of dihydrolipoamide succinyltransferase isolated without an expression tag and in this novel crystal form. [ABSTRACT FROM AUTHOR]

The conversion of phenylglycinonitrile (2-aminophenylacetonitrile) by Escherichia coli strains was studied, which recombinantly expressed the arylacetonitrilase (NitA) from Pseudomonas fluorescens EBC191 and different nitrilase variants with altered reaction specificities. The whole-cell catalysts which formed the wild-type nitrilase converted (R,S)-phenylglycinonitrile preferentially to (S)-phenylglycine with a low degree of enantioselectivity. A recombinant strain which formed a variant of NitA produced mainly (S)-phenylglycine amide from (R,S)-phenylglycinonitrile and a second variant showed an almost complete enantioconversion and produced (R)-phenylglycine and left (S)-phenylglycinonitrile. The microbial-produced (S)-phenylglycinonitrile was used to study the chemical racemisation of (S)-phenylglycinonitrile at alkaline pH values in order to establish a dynamic kinetic resolution of the substrate. Subsequently, the conversion of (R,S)-phenylglycinonitrile by the whole-cell catalysts was studied at a pH of 10.8 which allowed a sufficient racemisation rate of phenylglycinonitrile. Surprisingly, under these conditions, strongly increased amounts of (S)-phenylglycine were formed by the recombinant E. coli cells expressing the amide-forming nitrilase variant. The aminopeptidase PepA from E. coli was identified by the construction of a deletion mutant and subsequent complementation as responsible amidase activity, which converted (S)-phenylglycine amide to (S)-phenylglycine. [ABSTRACT FROM AUTHOR]

A disposable acetaminophen biosensor based on inkjet‐printed CNT electrodes (IJPCNT) modified with amidase/cerium dioxide@graphene nanoribbons composite was developed (ACeO2@GNR/IJPCNT). The enzyme amidase A was used for the first time as a recognition element. Inkjet‐printed CNT electrodes served as a basis for the construction of a biosensor that enables droplet detection using 5 μL sample volume. The biosensor showed high selectivity, sensitivity, a low detection limit of 0.18 μM and a wide working linear range from 1 to 100 μM. The proposed approach allows fast and reliable detection of acetaminophen in biological fluids with negligible matrix effect and remarkable reproducibility. [ABSTRACT FROM AUTHOR]

Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. In this study, a robust nitrilase from Arabis alpina (AaNIT) was screened and engineered. The N258D mutant was obtained with high catalytic activity and excellent enantioselectivity (E > 300) towards IBSN at a high substrate concentration of 100 g L−1. Byproduct (S)-3-cyano-5-methyl hexanoic amide ((S)-CMHM) was detected and identified for the first time during the catalytic process. By employing a feasible one-pot bienzymatic cascade of mutant N258D and amidase from Pantoea sp. (Pa-Ami) expressed separately in recombinant Escherichia coli cells, the byproduct (S)-CMHM was eliminated and (S)-CMHA was obtained with a conversion of 45.0% and eep of 99.3%. These results provided the novel plant-derived nitrilase as a promising biocatalyst for (S)-CMHA biosynthesis and demonstrated the feasibility of one-pot bienzymatic cascade reaction for large-scale production of the pregabalin precursor. [ABSTRACT FROM AUTHOR]

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electron effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from Pantoea sp. (Pa-Ami) was designed and engineered for improving of catalytic properties. Single mutant G175A and double mutant G175A/A305T exhibited 3.2- and 3.7-fold improvement in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased with kcat/Km values 3.1 and 10.0 times higher than that of the wild-type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of chloronicotinamide was shortened in the mutant G175A, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the double mutant G175A/A305T as biocatalyst, a maximum of 1220 mM of 2-chloronicotinic acid was produced with a 94% conversion and the space-time yield reached as high as 575 gproduct d-1. These results provide not only a novel robust biocatalyst for the production of chloronicotinic acid but also new insights into amidase structure-function relationships. [ABSTRACT FROM AUTHOR]

Catalyzed by an amidase‐containing Rhodococcus erythropolis AJ270 microbial whole cell catalyst in neutral phosphate buffer at 30 °C, desymmetric hydrolysis of a series of prochiral 3‐aryl and 3‐arylmethylglutaramides efficiently afforded 3‐substituted glutaric acid monoamides in up to 95% yield and >99.5% ee. Even far away from the reaction site, the substituents on the aryl still have a significant effect on the catalytic activity and enantioselectivity and different remote substituent effect was observed for the two types of substrates. The synthetic application of biocatalytic desymmetrization was demonstrated by the facile transformation of the obtained enantiopure (R)‐3‐substituted 4‐carbamoylbutanoic acid products to chiral dihydroquinolinone and δ‐lactone compounds. [ABSTRACT FROM AUTHOR]

• Four OTA and OTB-degrading enzymes were purified from the ML17 strain. • These four enzymes had a degradation rate of 100% for OTA and OTB. • These enzymes hydrolyze OTA and OTB into the low toxicity product OTα and OTβ respectively. Ochratoxin A (OTA) and Ochratoxin B (OTB) co-contaminate many types of agricultural products. Screening enzymes that degrade both OTA and OTB has significance in food safety. In this study, four novel OTA and OTB degrading enzymes, namely Bn OTase1, Bn OTase2, Bn OTase3, and Bn OTase4, were purified from the metabolites of the Brevundimonas naejangsanensis ML17 strain. These four enzymes hydrolyzed OTA into OTα and hydrolyzed OTB into OTβ. Bn OTase1, Bn OTase2, Bn OTase3, and Bn OTase4 have the apparent K m values for hydrolyzing OTA of 19.38, 0.92, 12.11, 1.09 μmol/L and for hydrolyzing OTB of 0.76, 2.43, 0.60, 0.64 μmol/L respectively. OTα and OTβ showed no significant cytotoxicity to HEK293 cells, suggesting that these enzymes mitigate the toxicity of OTA and OTB. The discovery of the novel OTA and OTB degrading enzymes enriches the research on ochratoxin control and provides objects for protein rational design. [ABSTRACT FROM AUTHOR]

Iprodione [3-(3,5-dichlorophenyl) N-isopropyl-2,4-dioxoimidazolidine-1- carboxamide] is a highly effective broad-spectrum dicarboxamide fungicide. Several bacteria with iprodione-degrading capabilities have been reported; however, the enzymes and genes involved in this process have not been characterized. In this study, an iprodione-degrading strain, Paenarthrobacter sp. strain YJN-5, was isolated and characterized. Strain YJN-5 degraded iprodione through the typical pathway, with hydrolysis of its N-1 amide bond to N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine as the initial step. The ipaH gene, encoding a novel amidase responsible for this step, was cloned from strain YJN-5 by the shotgun method. IpaH shares the highest similarity (40%) with an indoleacetamide hydrolase (IAHH) from Bradyrhizobium diazoefficiens USDA 110. IpaH displayed maximal enzymatic activity at 35°C and pH 7.5, and it was not a metalloamidase. The kcat and Km of IpaH against iprodione were 22.42 s-1 and 7.33 µM, respectively, and the catalytic efficiency value (kcat/Km) was 3.09 µM-1 s-1. IpaH has a Ser-Ser-Lys motif, which is conserved among members of the amidase signature family. The replacement of Lys82, Ser157, and Ser181 with alanine in IpaH led to the complete loss of enzymatic activity. Furthermore, strain YJN-5M lost the ability to degrade iprodione, suggesting that ipaH is the only gene responsible for the initial iprodione degradation step. The ipaH gene could also be amplified from another previously reported iprodione-degrading strain, Microbacterium sp. strain YJN-G. The sequence similarity between the two IpaHs at the amino acid level was 98%, indicating that conservation of IpaH exists in different strains. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is considered as one of the key competition strategies by injecting toxic effectors for intestinal pathogens to acquire optimal colonization in host gut, a microenviroment with high-density polymicrobial community where bacteria compete for niches and resources. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea in human and animals, widely encode T6SS clusters in their genomes. In this report, we first identified VT1, a novel amidase effector in ETEC, significantly hydrolyzed D-lactyl-L-Ala crosslinks between N -acetylmuramoyl and L-Ala in peptidoglycan. Further study showed that the VT1/VTI1 effector/immunity pair is encoded within a typical vgrG island, and plays a critical role for the successful establishment of ETEC in host gut. Numerous putative effectors with diverse toxin domains were found by retrieving vgrG islands in pathogenic E. coli , and designated as VT modules. Therein, VT5, a lysozyme-like effector widely encoded in ETEC, was confirmed to effectively kill adjacent cells, suggesting that VT toxin modules may be critical for pathogenic E. coli to seize a significantly competitive advantage for optimal intestinal colonization. To expand our analyses for large-scale search of VT antibacterial effectors based on vgrG island, >200 predicted effectors from 20 bacterial species were found and classified into 11 predicted toxins. This work reports a new retrieval strategy for screening T6SS effectors, and provides an example how pathogenic bacteria antagonize and displace commensal microbiome to successfully colonize in the host niches through a T6SS-dependent manner. [ABSTRACT FROM AUTHOR]

Summary: The essential stages of bacterial cell separation are described as the synthesis and hydrolysis of septal peptidoglycan (PG). The amidase, AmiC, which cleaves the peptide side‐chains linked to the glycan strands, contributes critically to this process and has been studied extensively in model strains of Escherichia coli. However, insights into the contribution of this protein to other processes in the bacterial cell have been limited. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogen that causes black rot disease in many economically important plants. We investigated how AmiC and LytM family regulators, NlpD and EnvC, contribute to virulence and cell separation in this organism. Biochemical analyses of purified AmiC demonstrated that it could hydrolyse PG and its activity could be potentiated by the presence of the regulator NlpD. We also established that deletion of the genes encoding amiC1 or nlpD led to a reduction in virulence as well as effects on colony‐forming units and cell morphology. Moreover, further genetic and biochemical evidence showed that AmiC1 and NlpD affect the secretion of type III effector XC3176 and hypersensitive response (HR) induction in planta. These findings indicate that, in addition to their well‐studied role(s) in cell separation, AmiC and NlpD make an important contribution to the type III secretion (T3S) and virulence regulation in this important plant pathogen. [ABSTRACT FROM AUTHOR]

Bacterial cell division is a complex process brought about by the coordinated action of multiple proteins. Separation of daughter cells during the final stages of division involves cleavage of new cell wall laid down at the division septum. In E. coli, this process is governed by the action of N-acetylmuramoyl-L-alanine amidases AmiA/B/C, which are regulated by their LytM activators EnvC and NlpD. While much is known about the regulation of septum cleavage in E. coli, the mechanism of daughter cell separation is not clear in Caulobacter crescentus, a dimorphic crescent-shaped bacterium. In this work, we characterized the role of AmiC, the only annotated amidase in C. crescentus. AmiC from C. crescentus is functional in E. coli and restores cell separation defects seen in E. coli amidase mutants, suggesting that AmiC has septum splitting activity. The medial localization of AmiC was independent of DipM, an LytM domain-containing endopeptidase. Our results indicate that enzymatic activity is essential for medial recruitment of AmiC. Overexpression of AmiC causes cell separation defects and formation of chains. Finally, overexpression of AmiC in cells inhibited for cell division leads to lysis. Collectively, our findings reveal that regulation of daughter cell separation in C. crescentus differs from that of E. coli and can serve as a model system to study bacterial cytokinesis. [ABSTRACT FROM AUTHOR]

Background Characterization of partially collapsed protein conformations at atomic level is a daunting task due to their inherent flexibility and conformational heterogeneity. T7 bacteriophage endolysin (T7L) is a single-domain amidase that facilitates the lysis of Gram-negative bacteria. T7L exhibits a pH-dependent structural transition from native state to partially folded (PF) conformation. In the pH range 5–3, T7L PF states display differential ANS binding characteristics. Methods CD, fluorescence, NMR spectroscopy and lysis assays were used to investigate the structure-stability- dynamics relationships of T7L PF conformations. Results Structural studies indicated a partial loss of secondary/tertiary structures compared to its native state. The loss in the tertiary structure and the hydrophobic core opening increases upon decrease of pH from 5 to 3. Thermal denaturation experiments delineated that the pH 5 conformation is thermally irreversible in contrast to pH 3, depicting that hydrophobic core opening is essential for thermal reversibility. Further, urea dependent unfolding features of PF state at pH 5 and 4 evidenced for a collapsed conformation at intermediate urea concentrations. Residue level studies revealed that α1-helix and β3-β4 segment of T7L are the major contributors for such a structural collapse and inherent dynamics. Conclusions The results suggested that the low pH PF states of T7L are heterogeneous and exhibits differential structural, unfolding, thermal reversibility, and dynamic features. General significance Unraveling the structure-stability characteristics of different endolysin conformations is essential for designing novel chimeric and engineered phage endolysins as broadband antimicrobial agents over a varied pH range. [ABSTRACT FROM AUTHOR]

Peptidoglycan recognition proteins (PGRPs) are indispensable molecules in innate immunity due to their prominent function in sensing and eliminating invading microorganisms. In the present study, a short type PGRP from razor clam Solen grandis (SgPGRP-S1) was recombinantly expressed and purified to investigate its potential function in innate immunity. As a pattern recognition receptor, recombinant SgPGRP-S1 (rSgPGRP-S1) specifically bind Lys-type and Dap-type peptidoglycan in vitro , but not lipopolysaccharide or β-glucan. The peptidoglycan binding ability of rSgPGRP-S1 resulted in significant agglutination activity against Gram-negative Escherichia coli and Listonella anguillarum , as well as Gram-positive Micrococcus luteus . Furthermore, rSgPGRP-S1 was bactericidal, significantly suppressing the growth of both E. coli and Gram-positive Staphylococcus aureus . The protein also exhibited strong amidase activity and degraded bacterial peptidoglycan in the presence of Zn 2+ , suggesting amidase activity might contribute to SgPGRP-S1 antibacterial activity. These results indicate SgPGRP-S1 is multifunctional in innate immunity, mediating both immune recognition and bacteria elimination. [ABSTRACT FROM AUTHOR]

2-Chloronicotinic acid (2-CA) is an important building block for a series of agrochemicals and pharmaceuticals. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is one of the most attractive approaches for 2-CA production. However, development of the bioprocess was plagued by low activity of amidase for 2-chloronicotinamide. In this work, an amidase signature (AS) family amidase from Pantoea sp. ( Pa -Ami), with superior activity for nicotinamide and its chlorinated derivatives, was exploited and characterized. Kinetic analysis and molecular docking clearly indicated that chlorine substitution in the pyridine ring of nicotinamide, especially the substitution at 2-position led to a dramatic decrease of Pa -Ami activity. The productivity of the bioprocess was significantly improved using fed-batch mode at low reaction temperature and 2-CA was produced as high as 370 mM with a substrate conversion of 94.2%. These results imply that Pa -Ami is potentially promising biocatalyst for industrial production of 2-CA. [ABSTRACT FROM AUTHOR]

The herbicide propanil and its major metabolite 3,4-dichloroaniline (3,4-DCA) are difficult to biodegrade and pose great health and environmental risks. However, studies on the sole or synergistic mineralization of propanil by pure cultured strains are limited. A two-strain consortium (Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34), obtained from a swep-mineralizing enrichment culture that can synergistically mineralize propanil, has been previously reported. Here, another propanil degradation strain, Bosea sp. P5, was successfully isolated from the same enrichment culture. A novel amidase, PsaA, responsible for initial propanil degradation, was identified from strain P5. PsaA shared low sequence identity (24.0–39.7 %) with other biochemically characterized amidases. PsaA exhibited optimal activity at 30 °C and pH 7.5 and had k cat and K m values of 5.7 s-1 and 125 μM, respectively. PsaA could convert the herbicide propanil to 3,4-DCA but exhibited no activity toward other herbicide structural analogs. This catalytic specificity was explained by using propanil and swep as substrates and then analyzed by molecular docking, molecular dynamics simulation and thermodynamic calculations, which revealed that Tyr138 is the key residue that affects the substrate spectrum of PsaA. This is the first propanil amidase with a narrow substrate spectrum identified, providing new insights into the catalytic mechanism of amidase in propanil hydrolysis [Display omitted] • An efficient propanil-transforming strain, P5, of the genus Bosea was isolated for the first time. • A new synergistic artificial consortium for propanil mineralization was successfully established. • The novel propanil amidase gene psaA was cloned, and PsaA was characterized. • Tyr138 is the key residue that affects the substrate specificity of PsaA. [ABSTRACT FROM AUTHOR]

Worldwide use of benzonitrile herbicides has caused a contamination hazard for groundwater, and their removal has attracted increasing attention. Microbial degradation has been considered as a major route of removing toxic nitriles from the environment. However, the process is very inefficient for degrading benzonitrile herbicides by natural microbes. In this study, a recombinant microbial cell was constructed to degrade benzonitrile herbicides by co-expression of nitrile hydratase and amidase in Escherichia coli . Both enzymes were functionally over-expressed in the cytoplasm of E. coli . The NHase activities of the cell reactor on dichlobenil and ioxynil were 15.4 and 21.3 U/mg dry cell weigh (DCW), respectively. And the amidase activities on 2,6-dichlorobenzamide and 3,5-iodo-4-hydroxybenzamide were 8.3 and 13.6 U/mg DCW, respectively. Furthermore, the degradation of dichlobenil and ioxynil was investigated using the recombinant cell reactor. The degradation process suggested that dichlobenil and ioxynil were degraded to corresponding carboxylic acids via nitrile hydratase-amidase pathway, and the intermediate amides (2,6-dichlorobenzamide and 3,5-iodo-4-hydroxybenzamide) did not accumulate in the reaction mixture. The degradation rates of dichlobenil and ioxynil were 43 and 185 mg/g DCW/h, respectively. The recombinant E. coli cell reactor was observed to be promising catalysts for the bioremediation of wastewater containing benzonitrile herbicides. [ABSTRACT FROM AUTHOR]

Recently, giant reed(Arundo donaxL.) has emerged as a useful bioresource capable of coping under harsh abiotic stress conditions produced by exposure to heavy metals, drought and salinity as evidenced by significant biomass production. The current study was undertaken to determine the expression of critical genes involved in resisting oxidative stress and maintaining normal cellular functions. The critical genes examined included glutathione reductase (GR) that prevents oxidative damage of membranes, carotenoid hydroxylase (CR), transcription factor (bHLH) and amidase involved in biosynthesis of indole-3-acetic acid following exposure to chromium (Cr) stress at concentrations of 0, 33.5, 67, 133 or 268 mg/L in giant reed. TheS19gene was used as internal control. It was not possible to extract RNA from severely stressed giant plant leaves at 133 and 268 mg/L Cr. Therefore, this investigation focused on gene expression in the presence of at 0, 33.5 or 67 mg/L Cr. Following Cr-mediated stress, gene expression of GR and CR was increased at 33.5 mg/L but reduced at 67 mg/L. The transcription factor, bHLH, gene expression remained unchanged. Amidase gene expression was elevated at 67 mg/L Cr. Data suggested that over expression of amidase and GR genes at high Cr exposure might increase growth and biomass of giant reed as well as enhance tolerance. These results indicated that giant reed tolerated the presence of Cr stress due to over expression of amidase and GR genes. It would appear that giant reed may serve a beneficial role for phytoremediation of Cr contaminated wastewater and soils at concentrations below 100 mg/kg. [ABSTRACT FROM AUTHOR]

The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ~35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria. [ABSTRACT FROM AUTHOR]

Summary Bacterial cell wall proteins play crucial roles in cell survival, growth, and environmental interactions. In Gram-positive bacteria, cell wall proteins include several types that are non-covalently attached via cell wall binding domains. Of the two conserved surface-layer (S-layer)-anchoring modules composed of three tandem SLH or CWB2 domains, the latter have so far eluded structural insight. The crystal structures of Cwp8 and Cwp6 reveal multi-domain proteins, each containing an embedded CWB2 module. It consists of a triangular trimer of Rossmann-fold CWB2 domains, a feature common to 29 cell wall proteins in Clostridium difficile 630. The structural basis of the intact module fold necessary for its binding to the cell wall is revealed. A comparison with previously reported atomic force microscopy data of S-layers suggests that C. difficile S-layers are complex oligomeric structures, likely composed of several different proteins. [ABSTRACT FROM AUTHOR]

Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase ( Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure ( R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co, with a specific activity of 753.5 U/mg, K value of 24.73 mM, and k /K value of 22.47 mM s. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess ( ee ) of 95 % within 10 min ( E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of ( R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. [ABSTRACT FROM AUTHOR]

In this study, an amidohydrolase activity of amidase in whole cells ofRhodococcussp. MTB5 has been used for the biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid, nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized against benzamide. The enzyme exhibited optimum activity in 50 mM potassium phosphate buffer pH 7.0. The optimum temperature and substrate concentrations for this enzyme were 50 °C and 50 mM, respectively. The enzyme was quite stable for more than 6 h at 30 °C. It showed substrate specificity against different amides, including aliphatic, aromatic and heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50 mM each of benzamide, nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120 min, respectively, using 5 mg dry cell mass (DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44% achieved by whole cells. The amidase in whole cells can withstand as high as 383 mM concentration of product in a reaction mixture and above which it undergoes product feedback inhibition. The results of this study suggest thatRhodococcussp. MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value. [ABSTRACT FROM PUBLISHER]

Monospecies and mixed bacterial biofilms of Rhodococcus ruber gt1, Pseudomonas fluorescens C2, Alcaligenes faecalis 2, and Rh. erythropolis 11-2 were obtained during growth in presence of the carriers. The transformation of aliphatic and aromatic nitriles by the biofilms of nitrile-hydrolyzing bacteria, as well as the growth dynamics of Rh. ruber gt1 and P. fluorescens C2 biofilms and their resistance to toxic substrates and products of nitrile hydrolysis, were studied. It was shown that the P. fluorescens C2 biofilm mass and total ATP content reached the maxima after 1 day of growth, whereas Rh. ruber gt1 reached them after 3-4 days of cultivation. The biofilms of Rh. ruber gt1 and P. fluorescens C2 were more resistant to the effects of high concentrations of acrylamide and acrylonitrile and had a greater adaptive capacity than planktonic cells. [ABSTRACT FROM AUTHOR]