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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]

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]

Nitrile-containing insecticides can be converted into their amide derivatives by Pseudaminobacter salicylatoxidans. N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) is converted to 4-(trifluoromethyl) nicotinoyl glycine (TFNG) using nitrile hydratase/amidase. However, the amidase that catalyzes this bioconversion has not yet been fully elucidated. In this study, it was discovered that flonicamid (FLO) is degraded by P. salicylatoxidans into the acid metabolite TFNG via the intermediate TFNG-AM. A half-life of 18.7 h was observed for P. salicylatoxidans resting cells, which transformed 82.8% of the available FLO in 48 h. The resulting amide metabolite, TFNG-AM, was almost all converted to TFNG within 19 d. A novel amidase-encoding gene was cloned and overexpressed in Escherichia coli. The enzyme, PmsiA, hydrolyzed TFNG-AM to TFNG. Despite being categorized as a member of the amidase signature enzyme superfamily, PsmiA only shares 20–30% identity with the 14 previously identified members of this family, indicating that PsmiA represents a novel class of enzyme. Homology structural modeling and molecular docking analyses suggested that key residues Glu247 and Met242 may significantly impact the catalytic activity of PsmiA. This study contributes to our understanding of the biodegradation process of nitrile-containing insecticides and the relationship between the structure and function of metabolic enzymes. [ABSTRACT FROM AUTHOR]

In recent years, biocatalysts have emerged as crucial tool in organic synthesis, particularly for the production of drug intermediates and precursors, e.g., the synthesis of hydroxamic acids. Traditionally, hydroxamic acids were synthesized using organic chemistry methods. However, with the growing emphasis on sustainable and environment‐friendly practices, the chemical industry has increasingly turned towards green synthesis approaches. The significance of hydroxamic acids in medicinal chemistry has also contributed to the changing trends. Following the approval of certain hydroxamic acids as histone deacetylase (HDAC) inhibitors for cancer treatment by the Food and Drug Administration (US‐FDA), there has been a renewed focus on their synthesis and the development of derivatives with improved properties. As an alternative route, amidases have emerged as promising biocatalysts for hydroxamic acid synthesis through their acyltransferase activity. Recent advancements in the synthesis approaches for hydroxamic acids are reviewed. The biocatalytic routes are explored, emphasizing the use of amidases and their acyltransferase activity. The scope and potential applications of this chemoenzymatic approach in synthesizing various hydroxamic acids and their derivatives are discussed. Such advancements have the potential to revolutionize the production of these important compounds, making the synthesis process more sustainable, efficient, and economically viable. [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]

The fungicide iprodione (IPR) (3-(3,5-dichlorophenyl) N-isopropyl-2,4-dioxoimidazolidine-1-carboxamide) is a highly toxic compound. Although IPR has been restricted, it is still being applied in many places around the world, constituting an environmental risk. The biodegradation of IPR is an attractive option for reducing its residues. In this study, we isolated thirteen IPR-tolerant bacteria from a biopurification system designed to treat pesticides. A study of biodegradation using different strains was comparatively evaluated, and the best degradation rate of IPR was presented by Achromobacter sp. C1 with a half-life (T1/2) of 9 days. Based on a nano-LC-MS/MS analysis for the strains, proteins solely expressed in the IPR treatment were identified by highlighting the strain Achromobacter sp. C1, with 445 proteins primarily involved in the biosynthesis of secondary metabolites and microbial metabolism in diverse environments. Differentially expressed protein amidases were involved in six metabolic pathways. Interestingly, formamidase was inhibited while other cyclases, i.e., amidase and mandelamide hydrolase, were overexpressed, thereby minimizing the effect of IPR on the metabolism of strain C1. The dynamic changes in the protein profiles of bacteria that degrade IPR have been poorly studied; therefore, our results offer new insight into the metabolism of IPR-degrading microorganisms, with special attention paid to amidases. [ABSTRACT FROM AUTHOR]

Nitrile-containing insecticides can be converted into their amide derivatives by Pseudaminobacter salicylatoxidans. N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) is converted to 4-(trifluoromethyl) nicotinoyl glycine (TFNG) using nitrile hydratase/amidase. However, the amidase that catalyzes this bioconversion has not yet been fully elucidated. In this study, it was discovered that flonicamid (FLO) is degraded by P. salicylatoxidans into the acid metabolite TFNG via the intermediate TFNG-AM. A half-life of 18.7 h was observed for P. salicylatoxidans resting cells, which transformed 82.8% of the available FLO in 48 h. The resulting amide metabolite, TFNG-AM, was almost all converted to TFNG within 19 d. A novel amidase-encoding gene was cloned and overexpressed in Escherichia coli. The enzyme, PmsiA, hydrolyzed TFNG-AM to TFNG. Despite being categorized as a member of the amidase signature enzyme superfamily, PsmiA only shares 20–30% identity with the 14 previously identified members of this family, indicating that PsmiA represents a novel class of enzyme. Homology structural modeling and molecular docking analyses suggested that key residues Glu247 and Met242 may significantly impact the catalytic activity of PsmiA. This study contributes to our understanding of the biodegradation process of nitrile-containing insecticides and the relationship between the structure and function of metabolic enzymes.

ABSTRACT The peptidoglycan layer is a defining characteristic of bacterial cells, providing them with structural support and osmotic protection. In Escherichia coli, this layer is linked to the outer membrane via the abundant membrane-anchored protein Lpp, known as Braun’s lipoprotein, with LD-transpeptidases LdtA, LdtB, and LdtC catalyzing the attachment. However, one distinctive member of the YkuD-type transpeptidase family, LdtF (recently renamed DpaA), carries out the opposite reaction of detaching Lpp from the peptidoglycan layer. In this study, we report the crystal structure of DpaA, which reveals the enzyme’s ability to cleave, rather than form, the Lpp-peptidoglycan linkage. Assays with purified peptidoglycan-Lpp as the substrate and chemically synthesized compounds suggest that DpaA’s shallow L-shaped active site can only accommodate and cleave the peptidoglycan-Lpp cross-link with a constrained conformation. This study provides insights into how homologous Ldt enzymes can perform opposing chemical reactions. IMPORTANCE Cross-linking reaction of Braun's lipoprotein (Lpp) to peptidoglycan (PG) is catalyzed by some members of the YkuD family of transpeptidases. However, the exact opposite reaction of cleaving the Lpp-PG cross-link is performed by DpaA, which is also a YkuD-like protein. In this work, we determined the crystal structure of DpaA to provide the molecular rationale for the ability of the transpeptidase-like protein to cleave, rather than form, the Lpp-PG linkage. Our findings also revealed the structural features that distinguish the different functional types of the YkuD family enzymes from one another.

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]

The reverse genetic approach has uncovered indole synthase (INS) as the first enzyme in the tryptophan (trp)-independent pathway of IAA synthesis. The importance of INS was reevaluated suggesting it may interact with tryptophan synthase B (TSB) and therefore involved in the trp-dependent pathway. Thus, the main aim of this study was to clarify the route of INS through the analysis of Arabidopsis genome. Analysis of the top 2000 co-expression gene lists in general and specific conditions shows that TSA is strongly positively co-expressed with TSB in general, hormone, and abiotic conditions with mutual ranks of 89, 38, and 180 respectively. Moreover, TSA is positively correlated with TSB (0.291). However, INS was not found in any of these coexpressed gene lists and negatively correlated with TSB (− 0.046) suggesting unambiguously that these two routes are separately and independently operated. So far, the remaining steps in the INS pathway have remained elusive. Among all enzymes reported to have a role in IAA synthesis, amidase was found to strongly positively co-expressed with INS in general and light conditions with mutual ranks of 116 and 141 respectively. Additionally, amidase1 was found to positively correlate with INS (0.297) and negatively coexpressed with TSB concluding that amidase may exclusively involve in the trp-independent pathway. [ABSTRACT FROM AUTHOR]

Triclocarban (TCC) and its metabolite, 3,4-dichloroaniline (DCA), are classified as emerging organic contaminants (EOCs). Significant concerns arise from water and soil contamination with TCC and its metabolites. These concerns are especially pronounced at high concentrations of up to approximately 20 mg/kg dry weight, as observed in wastewater treatment plants (WWTPs). Here, a TCC-degrading co-culture system comprising Rhodococcus rhodochrous BX2 and Pseudomonas sp. LY-1 was utilized to degrade TCC (14.5 mg/L) by 85.9% in 7 days, showing improved degradation efficiency compared with monocultures. A combination of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), genome sequencing, transcriptomic analysis, and quantitative reverse transcription-PCR (qRT-PCR) was performed. Meanwhile, through the combination of further experiments involving heterologous expression and gene knockout, we proposed three TCC metabolic pathways and identified four key genes (tccG , tccS , phB , phL) involved in the TCC degradation process. Moreover, we revealed the internal labor division patterns and connections in the co-culture system, indicating that TCC hydrolysis products were exchanged between co-cultured strains. Additionally, mutualistic cooperation between BX2 and LY-1 enhances TCC degradation efficiency. Finally, phytotoxicity assays confirmed a significant reduction in the plant toxicity of TCC following synergistic degradation by two strains. The in-depth understanding of the TCC biotransformation mechanisms and microbial interactions provides useful information for elucidating the mechanism of the collaborative biodegradation of various contaminants. [Display omitted] • An efficient degradation system for triclocarban was established by two bacterial strains. • The efficient degradation system degraded 85.9% of 14.5 mg/L TCC. • Further degradation of the intermediate 3, 4 - dichloroaniline from TCC was achieved. • The internal labor division was revealed by three metabolic pathways in the co-culture system. [ABSTRACT FROM AUTHOR]

The fungicide iprodione (IPR) (3-(3,5-dichlorophenyl) N-isopropyl-2,4-dioxoimidazolidine-1-carboxamide) is a highly toxic compound. Although IPR has been restricted, it is still being applied in many places around the world, constituting an environmental risk. The biodegradation of IPR is an attractive option for reducing its residues. In this study, we isolated thirteen IPR-tolerant bacteria from a biopurification system designed to treat pesticides. A study of biodegradation using different strains was comparatively evaluated, and the best degradation rate of IPR was presented by Achromobacter sp. C1 with a half-life (T1/2) of 9 days. Based on a nano-LC-MS/MS analysis for the strains, proteins solely expressed in the IPR treatment were identified by highlighting the strain Achromobacter sp. C1, with 445 proteins primarily involved in the biosynthesis of secondary metabolites and microbial metabolism in diverse environments. Differentially expressed protein amidases were involved in six metabolic pathways. Interestingly, formamidase was inhibited while other cyclases, i.e., amidase and mandelamide hydrolase, were overexpressed, thereby minimizing the effect of IPR on the metabolism of strain C1. The dynamic changes in the protein profiles of bacteria that degrade IPR have been poorly studied; therefore, our results offer new insight into the metabolism of IPR-degrading microorganisms, with special attention paid to amidases.

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.

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A versatile biocatalytic desymmetric method for efficiently accessing enantiopure cis-3,4-disubstituted pyrrolidines was developed. Catalyzed by amidase-containing E. coli whole cells, a series of meso pyrrolidine-2,5-dicarboxamides were hydrolyzed to obtain 4-carbamoylpyrrolidine-3-carboxylic acid derivatives in 47%–95% yields and 62% ∼ >99.5% ee values under mild condition. The catalytic efficiency and enantioselectivity are related to the substituents in the phenyl ring. The enzyme-substrate binding mode is established and the high enantioselectivity of amidase is revealed by MD simulations. The improvement of biocatalytic efficiency has been preliminarily explored through protein engineering.

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]

The high mortality rate associated with Listeria monocytogenes and its ability to adapt to the harsh conditions employed in food processing has ensured that this pathogen remains a serious problem in the ready-to-eat food sector. Bacteriophage-derived enzymes can be applied as biocontrol agents to target specific foodborne pathogens. We investigated the ability of a listeriophage endolysin and derivatives thereof, fused to polyhydroxyalkanoate bionanoparticles (PHA_BNPs), to lyse and inhibit the growth of L. monocytogenes. Turbidity reduction assays confirmed the lysis of L. monocytogenes cells at 37 °C upon addition of the tailored BNPs. The application of BNPs also resulted in the growth inhibition of L. monocytogenes. BNPs displaying only the amidase domain of the phage endolysin were more effective at inhibiting growth under laboratory conditions (37 °C, 3 × 107 CFU/mL) than BNPs displaying the full-length endolysin (89% vs. 83% inhibition). Under conditions that better represent those found in food processing environments (22 °C, 1 × 103 CFU/mL), BNPs displaying the full-length endolysin demonstrated a greater inhibitory effect compared to BNPs displaying only the amidase domain (61% vs. 54% inhibition). Our results demonstrate proof-of-concept that tailored BNPs displaying recombinant listeriophage enzymes are active inhibitors of L. monocytogenes. [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]

Abstract 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.

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]

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity. [ABSTRACT FROM AUTHOR]

Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO) is a new type of pyridinecarboxamide insecticide that exhibits particularly good efficacy in pest control. However, the extensive use of FLO in agricultural production poses environmental risks. Hence, its environmental behavior and degradation mechanism have received increasing attention. Microvirga flocculans CGMCC 1.16731 rapidly degrades FLO to produce the intermediate N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) and the end acid metabolite 4-(trifluoromethyl) nicotinol glycine (TFNG). This bioconversion is mediated by the nitrile hydratase/amidase system; however, the amidase that is responsible for the conversion of TFNG-AM to TFNG has not yet been reported. Here, gene cloning, overexpression in Escherichia coli and characterization of pure enzymes showed that two amidases—AmiA and AmiB—hydrolyzed TFNG-AM to TFNG. AmiA and AmiB showed only 20–30% identity to experimentally characterized amidase signature family members, and represent novel amidases. Compared with AmiA, AmiB was more sensitive to silver and copper ions but more resistant to organic solvents. Both enzymes demonstrated good pH tolerance and exhibited broad amide substrate specificity. Homology modeling suggested that residues Asp191 and Ser195 may strongly affect the catalytic activity of AmiA and AmiB, respectively. The present study furthers our understanding of the enzymatic mechanisms of biodegradation of nitrile-containing insecticides and may aid in the development of a bioremediation agent for FLO.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

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.

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]

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]

The high mortality rate associated with Listeria monocytogenes and its ability to adapt to the harsh conditions employed in food processing has ensured that this pathogen remains a serious problem in the ready-to-eat food sector. Bacteriophage-derived enzymes can be applied as biocontrol agents to target specific foodborne pathogens. We investigated the ability of a listeriophage endolysin and derivatives thereof, fused to polyhydroxyalkanoate bionanoparticles (PHA_BNPs), to lyse and inhibit the growth of L. monocytogenes. Turbidity reduction assays confirmed the lysis of L. monocytogenes cells at 37 °C upon addition of the tailored BNPs. The application of BNPs also resulted in the growth inhibition of L. monocytogenes. BNPs displaying only the amidase domain of the phage endolysin were more effective at inhibiting growth under laboratory conditions (37 °C, 3 × 107 CFU/mL) than BNPs displaying the full-length endolysin (89% vs. 83% inhibition). Under conditions that better represent those found in food processing environments (22 °C, 1 × 103 CFU/mL), BNPs displaying the full-length endolysin demonstrated a greater inhibitory effect compared to BNPs displaying only the amidase domain (61% vs. 54% inhibition). Our results demonstrate proof-of-concept that tailored BNPs displaying recombinant listeriophage enzymes are active inhibitors of L. monocytogenes.

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]

Objective: Cloning and secretory expression of an amidase from Kluyvera cryocrescens and characterization of its potential in preparation of chiral amino acids. Results: An amidase belonging to the Ntn-hydrolase superfamily was identified from Kluyvera cryocrescens ZJB-17005 (Kc-Ami). The maximum activity of Kc-Ami was observed at pH 8.5 and 55 °C. Remarkably, Kc-Ami showed an excellent enantioselectivity (99% ee) using rac-4-(hydroxy(methyl)phosphoryl)-2-(2-phenylacetamido) butanoic acid as substrate. Kc-Ami remained stable at pH 7.0–9.0 and exhibited prominent thermostability with a half-life time of 59.1, 47.4 and 20.4 h at 50, 55 and 60 °C, respectively. Kc-Ami could be appllied to synthesize chiral amino acids and its derivatives with excellent enantioselectivity (> 99% ee). The synthesized chiral amino acids could contain short or long side chain, and further the side chain could be replaced with –OH, –COOH or benzene ring. Conclusions: Kc-Ami exhibited remarkable thermostability and excellent enantioselectivity for synthesizing chiral amino acids and its derivatives. This specific characteristic provides great potential for industrial application in preparation of chiral amino acids and its derivatives. [ABSTRACT FROM AUTHOR]

ABSTRACT In Escherichia coli, FtsEX coordinates peptidoglycan (PG) synthesis and hydrolysis at the septum. It acts on FtsA in the cytoplasm to promote recruitment of septal PG synthetases and recruits EnvC, an activator of septal PG hydrolases, in the periplasm. Following recruitment, ATP hydrolysis by FtsEX is thought to regulate both PG synthesis and hydrolysis, but how it does this is not well understood. Here, we show that an ATPase mutant of FtsEX blocks septal PG synthesis similarly to cephalexin, suggesting that ATP hydrolysis by FtsEX is required throughout septation. Using mutants that uncouple the roles of FtsEX in septal PG synthesis and hydrolysis, we find that recruitment of EnvC to the septum by FtsEX, but not ATP hydrolysis, is required to promote cell separation when the NlpD-mediated cell separation system is present. However, ATP hydrolysis by FtsEX becomes necessary for efficient cell separation when the NlpD system is inactivated, suggesting that the ATPase activity of FtsEX is required for optimal activity of EnvC. Importantly, under conditions that suppress the role of FtsEX in cell division, disruption of the FtsEX-FtsA interaction delays cell separation, highlighting the importance of this interaction in coupling the cell separation system with the septal PG synthetic complex. IMPORTANCE Cytokinesis in Gram-negative bacteria requires coordinated invagination of the three layers of the cell envelope; otherwise, cells become sensitive to hydrophobic antibiotics and can even undergo cell lysis. In E. coli, the ABC transporter FtsEX couples the synthesis and hydrolysis of the stress-bearing peptidoglycan layer at the septum by interacting with FtsA and EnvC, respectively. ATP hydrolysis by FtsEX is critical for its function, but the reason why is not clear. Here, we find that in the absence of ATP hydrolysis, FtsEX blocks septal PG synthesis similarly to cephalexin. However, an FtsEX ATPase mutant, under conditions where it cannot block division, rescues ftsEX phenotypes as long as a partially redundant cell separation system is present. Furthermore, we find that the FtsEX-FtsA interaction is important for efficient cell separation.

L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme belonging to the peptidase S33 family, and is highly similar to prolyl aminopeptidases (PAP, EC. 3.4.11.5). Besides being an S-stereoselective character towards piperidine-based carboxamides, this enzyme also hydrolyses different L-amino acid amides, turning it into a potential biocatalyst within the Amidase Process. In this work, we report the characterization of L-proline amide hydrolase from Pseudomonas syringae (PsyPAH) together with the first X-ray structure for this class of L-amino acid amidases. Recombinant PsyPAH showed optimal conditions at pH 7.0 and 35 °C, with an apparent thermal melting temperature of 46 °C. The enzyme behaved as a monomer at the optimal pH. The L-enantioselective hydrolytic activity towards different canonical and non-canonical amino-acid amides was confirmed. Structural analysis suggests key residues in the enzymatic activity.

Acrylamide (AA) present in food is considered a harmful compound for humans, but it exerts an impact on microorganisms too. The aim of this study was to evaluate the impact of acrylamide (at conc. 0–10 µg/mL) on the growth of bacteria (Leuconostoc mesenteroides, Lactobacillus acidophilus LA-5) and yeasts (Saccharomyces cerevisiae, Kluyveromyces lactis var. lactis), which are used for food fermentation. Moreover, we decided to verify whether these microorganisms could utilise acrylamide as a nutritional compound. Our results proved that acrylamide can stimulate the growth of L. acidophilus and K. lactis. We have, to the best of our knowledge, reported for the first time that the probiotic strain of bacteria L. acidophilus LA-5 is able to utilise acrylamide as a source of carbon and nitrogen if they lack them in the environment. This is probably due to acrylamide degradation by amidases. The conducted response surface methodology indicated that pH as well as incubation time and temperature significantly influenced the amount of ammonia released from acrylamide by the bacteria. In conclusion, our studies suggest that some strains of bacteria present in milk fermented products can exert additional beneficial impact by diminishing the acrylamide concentration and hence helping to prevent against its harmful impact on the human body and other members of intestinal microbiota.