RESUMEN

The anti-diabetic drug metformin is one of the most widely prescribed medicines in the world. Together with its degradation product guanylurea, it is a major pharmaceutical pollutant in wastewater treatment plants and surface waters. An operon comprising two genes of the ureohydrolase family in Pseudomonas and Aminobacter species has recently been implicated in metformin degradation. However, the corresponding proteins have not been characterized. Here we show that these genes encode a Ni2+-dependent enzyme that efficiently and specifically hydrolyzes metformin to guanylurea and dimethylamine. The active enzyme is a heteromeric complex of α- and ß- subunits in which only the α-subunits contain the conserved His and Asp residues for the coordination of two Ni2+ ions in the active site. A crystal structure of metformin hydrolase reveals an α2ß4 stoichiometry of the hexameric complex, which is unprecedented in the ureohydrolase family. By studying a closely related but more widely distributed enzyme, we find that the putative predecessor specifically hydrolyzes dimethylguanidine instead of metformin. Our findings establish the molecular basis for metformin hydrolysis to guanylurea as the primary pathway for metformin biodegradation and provide insight into the recent evolution of ureohydrolase family proteins in response to an anthropogenic compound.

Asunto(s)

Metformina , Níquel , Metformina/metabolismo , Metformina/química , Níquel/metabolismo , Níquel/química , Ureohidrolasas/metabolismo , Ureohidrolasas/genética , Ureohidrolasas/química , Cristalografía por Rayos X , Dominio Catalítico , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Hidrólisis , Biodegradación Ambiental , Pseudomonas/enzimología , Pseudomonas/genética , Modelos Moleculares

RESUMEN

1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in the isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a five gene-cluster encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The γ-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. A glutamine synthetase-like (GS-like) enzyme (NpaA1) initiates 1NA glutamylation, and this enzyme exhibits a broad substrate selectivity toward a variety of anilines and naphthylamine derivatives. Structural analysis revealed that the aromatic residues in the 1NA entry tunnel and the V201 site in the large substrate-binding pocket significantly influence NpaA1's substrate preferences. The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites.

Asunto(s)

1-Naftilamina , Pseudomonas , Pseudomonas/enzimología , Pseudomonas/genética , Pseudomonas/metabolismo , Especificidad por Sustrato , 1-Naftilamina/análogos & derivados , 1-Naftilamina/metabolismo , Biodegradación Ambiental , Dioxigenasas/metabolismo , Dioxigenasas/genética , Dioxigenasas/química , Redes y Vías Metabólicas , Familia de Multigenes , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química

RESUMEN

The rising utilization of PLA/PBAT-ST20 presents potential ecological risks stemming from its casual disposal and incomplete degradation. To solve this problem, this study investigated the degradation capabilities of PLA/PBAT-ST20 by a co-culture system comprising two thermophilic bacteria, Pseudomonas G1 and Kocuria G2, selected and identified from the thermophilic phase of compost. Structural characterization results revealed that the strains colonized the PLA/PBAT-ST20's surface, causing holes and cracks, with an increase in the carbonyl index (CI) and polydispersity index (PDI), indicating oxidative degradation. Enzyme activity results demonstrated that the co-culture system significantly enhanced the secretion and activity of proteases and lipases, promoting the breakdown of ester bonds. LC-QTOF-MS results showed that various intermediate products were obtained after degradation, ultimately participating in the TCA cycle (ko00020), further completely mineralized. Additionally, after 15-day compost, the co-culture system achieved a degradation rate of 72.14 ± 2.1 wt% for PBAT/PLA-ST20 films, with a decrease in the abundance of plastic fragments of all sizes, demonstrating efficient degradation of PLA/PBAT-ST20 films. This study highlights the potential of thermophilic bacteria to address plastic pollution through biodegradation and emphasizes that the co-culture system could serve as an ideal solution for the remediation of PLA/PBAT plastics.

Asunto(s)

Biodegradación Ambiental , Técnicas de Cocultivo , Pseudomonas/metabolismo , Pseudomonas/enzimología , Poliésteres/metabolismo , Poliésteres/química , Redes y Vías Metabólicas , Plásticos Biodegradables/metabolismo , Plásticos Biodegradables/química , Microbiología del Suelo

RESUMEN

In seeding plants, biosynthesis of the phytohormone ethylene, which regulates processes including fruit ripening and senescence, is catalyzed by 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. The plant pathogen Pseudomonas savastanoi (previously classified as: Pseudomonas syringae) employs a different type of ethylene-forming enzyme (psEFE), though from the same structural superfamily as ACC oxidase, to catalyze ethylene formation from 2-oxoglutarate (2OG) in an arginine dependent manner. psEFE also catalyzes the more typical oxidation of arginine to give L-Δ1-pyrroline-5-carboxylate (P5C), a reaction coupled to oxidative decarboxylation of 2OG giving succinate and CO2. We report on the effects of C3 and/or C4 substituted 2OG derivatives on the reaction modes of psEFE. 1H NMR assays, including using the pure shift method, reveal that, within our limits of detection, none of the tested 2OG derivatives is converted to an alkene; some are converted to the corresponding ß-hydroxypropionate or succinate derivatives, with only the latter being coupled to arginine oxidation. The NMR results reveal that the nature of 2OG derivatization can affect the outcome of the bifurcating reaction, with some 2OG derivatives exclusively favoring the arginine oxidation pathway. Given that some of the tested 2OG derivatives are natural products, the results are of potential biological relevance. There are also opportunities for therapeutic or biocatalytic regulation of the outcomes of reactions catalyzed by 2OG-dependent oxygenases by the use of 2OG derivatives.

Asunto(s)

Proteínas Bacterianas , Etilenos , Ácidos Cetoglutáricos , Pseudomonas , Pseudomonas/enzimología , Pseudomonas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Etilenos/metabolismo , Etilenos/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Liasas/metabolismo , Liasas/química , Liasas/genética , Arginina/metabolismo , Arginina/química , Oxidación-Reducción

RESUMEN

Background/purposes: The continuously increasing carbapenem resistance within Enterobacterales and Pseudomonas poses a threat to public health, nevertheless, the molecular characteristics of which in southern China still remain limited. And carbapenemase identification is a key factor in effective early therapy of carbapenem-resistant bacteria infections. We aimed to determine the molecular characteristics of these pathogens and compare commercial combined disc tests (CDTs) with the modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) in detecting and distinguishing carbapenemases using whole genome sequencing (WGS). Methods: A total of 78 Enterobacterales, 30 Pseudomonas were obtained from two tertiary hospitals in southern China. Susceptibility tests were conducted using an automated VITEK2 compact system with confirmation via the Kirby-Bauer method. The WGS was conducted on all clinical isolates and the molecular characteristics were analyzed by screening the whole genome sequences. CDTs with or without cloxacillin, mCIM, and eCIM, were performed and compared by taking WGS results as the benchmark. Results: A total of 103 carbapenem non-susceptible and 5 carbapenem susceptible bacteria were determined, with Klebsiella pneumoniae (42.7%), Pseudomonas aeruginosa (23.3%) and Escherichia coli (18.4%) being most prevalent. Carbapenemase genes were detected in 58 (56.3%) of the 103 carbapenem-non-susceptible clinical isolates, including 46 NDM, 6 KPC, 3 IMP, 1 IPM+VIM,1NDM+KPC, and 1 OXA-181. Carbapenemase-producing isolates were detected more frequently in Enterobacterales (76.3%). Among K. pneumoniae, the major sequence types were st307 and st11, while among E. coli and P. aeruginosa, the most prevalent ones were st410 and st242 respectively. For carbapenemase detection in Enterobacterales, the mCIM method achieved 100.00% (95% CI, 92.13-100.00%) sensitivity and 94.44% (70.63-99.71%) specificity (kappa, 0.96); for Pseudomonas, detection sensitivity was 100% (5.46-100.00%), and 100% (84.50-100.00%) specificity (kappa, 0.65). Commercial CDT carbapenemase detection sensitivity for Enterobacterales was 96.49% (86.84-99.39%), and 95.24% (74.13-99.75%) specificity (kappa, 0.90); for Pseudomonas, carbapenemase detection sensitivity was 100.00% (5.46-100.00%) and 37.93% (21.30-57.64%) specificity (kappa, 0.04). When cloxacillin testing was added, CDT specificity reached 84.61% (64.27-94.95%). Conclusion: The molecular epidemiology of carbapenem-non-susceptible isolates from pediatric patients in Southern China exhibited distinctive characteristics. Both the mCIM-eCIM combination and CDT methods effectively detected and differentiated carbapenemases among Enterobacterales isolates, and the former performed better than CDT among Pseudomonas.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Pruebas de Sensibilidad Microbiana , Pseudomonas , Secuenciación Completa del Genoma , beta-Lactamasas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuenciación Completa del Genoma/métodos , beta-Lactamasas/genética , Humanos , Pseudomonas/genética , Pseudomonas/efectos de los fármacos , Pseudomonas/enzimología , Pseudomonas/aislamiento & purificación , China , Antibacterianos/farmacología , Enterobacteriaceae/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , Carbapenémicos/farmacología , Genoma Bacteriano , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Pseudomonas/microbiología , Fenotipo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/aislamiento & purificación

RESUMEN

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.

Asunto(s)

Heces , Hidrolasas , Tereftalatos Polietilenos , Streptomyces , Tereftalatos Polietilenos/metabolismo , Tereftalatos Polietilenos/química , Streptomyces/enzimología , Streptomyces/genética , Hidrolasas/metabolismo , Hidrolasas/genética , Hidrolasas/química , Humanos , Heces/microbiología , Pseudomonas/enzimología , Pseudomonas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderiales

RESUMEN

Marine environments harbor a plethora of microorganisms that represent a valuable source of new biomolecules of biotechnological interest. In particular, enzymes from marine bacteria exhibit unique properties due to their high catalytic activity under various stressful and fluctuating conditions, such as temperature, pH, and salinity, fluctuations which are common during several industrial processes. In this study, we report a new esterase (EstGoM) from a marine Pseudomonas sp. isolated at a depth of 1000 m in the Gulf of Mexico. Bioinformatic analyses revealed that EstGoM is an autotransporter esterase (type Va) and belongs to the lipolytic family II, forming a new subgroup. The purified recombinant EstGoM, with a molecular mass of 67.4 kDa, showed the highest hydrolytic activity with p-nitrophenyl octanoate (p-NP C8), although it was also active against p-NP C4, C5, C10, and C12. The optimum pH and temperature for EstGoM were 9 and 60 °C, respectively, but it retained more than 50% of its activity over the pH range of 7-11 and temperature range of 10-75 °C. In addition, EstGoM was tolerant of up to 1 M NaCl and resistant to the presence of several metal ions, detergents, and chemical reagents, such as EDTA and ß-mercaptoethanol. The enzymatic properties of EstGoM make it a potential candidate for several industrial applications.

Asunto(s)

Esterasas , Pseudomonas , Pseudomonas/enzimología , Pseudomonas/genética , Especificidad por Sustrato , Esterasas/metabolismo , Esterasas/genética , Esterasas/química , Concentración de Iones de Hidrógeno , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Temperatura , Estabilidad de Enzimas , Filogenia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Agua de Mar/microbiología

RESUMEN

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, ChdH114A exhibited catalytic activity with a kcat value of 1.07 s-1, ~ 5% of wild-type (WT) Chd, and a KM of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and ChdH114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2 Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on ChdH114A, Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented.

Asunto(s)

Histidina , Nitrilos , Pseudomonas , Pseudomonas/enzimología , Pseudomonas/metabolismo , Nitrilos/metabolismo , Nitrilos/química , Histidina/química , Histidina/metabolismo , Hidrólisis , Biocatálisis , Dominio Catalítico , Fungicidas Industriales/química , Fungicidas Industriales/metabolismo , Halogenación , Hidrolasas/metabolismo , Hidrolasas/química

RESUMEN

The protective mode of PostbioYDFF-3 (referred to as postbiotics) on the quality stability of refrigerated fillets was explored from the aspects of endogenous enzyme activity and the abundance of spoilage microorganisms. Compared to the control group, the samples soaked in postbiotics showed significant reductions in TVC, TVB-N and TBARS values by 39.6%, 58.6% and 25.5% on day 5, respectively. In addition, the color changes, biogenic amine accumulation and texture softening of the fish fillets soaked in postbiotics were effectively suppressed. Furthermore, the activity of endogenous enzyme activities was detected. The calpain activities were significantly inhibited (p < 0.05) after soaking in postbiotics, which declined by 23%. Meanwhile, high throughput sequencing analysis further indicated that the growth of spoilage microorganism such as Acinetobacter and Pseudomonas were suppressed. Overall, the PostbioYDFF-3 was suitable for preserving fish meat.

Asunto(s)

Bacterias , Carpas , Conservación de Alimentos , Alimentos Marinos , Animales , Alimentos Marinos/análisis , Alimentos Marinos/microbiología , Conservación de Alimentos/métodos , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/clasificación , Bacterias/enzimología , Refrigeración , Conservantes de Alimentos/farmacología , Carne/análisis , Carne/microbiología , Pseudomonas/enzimología , Pseudomonas/crecimiento & desarrollo

RESUMEN

OBJECTIVES: To characterize VIM-type metallo-ß-lactamase (MBL)-encoding genomic islands (GIs) in Pseudomonas aeruginosa and P. putida group isolates from Polish hospitals from 2001-2015/16. METHODS: Twelve P. aeruginosa and 20 P. putida group isolates producing VIM-like MBLs were selected from a large collection of these based on epidemiological and typing data. The organisms represented all major epidemic genotypes of these species spread in Poland with chromosomally located blaVIM gene-carrying integrons. The previously determined short-read sequences were complemented by long-read sequencing in this study. The comparative structural analysis of the GIs used a variety of bioinformatic tools. RESULTS: Thirty different GIs with blaVIM integrons were identified in the 32 isolates, of which 24 GIs from 26 isolates were integrative and conjugative elements (ICEs) of the clc family. These in turn were dominated by 21 variants of the GI2/ICE6441 subfamily with a total of 19 VIM integrons, each inserted in the same position within the ICE's Tn21-like transposon Tn4380. The three other ICEs formed a novel ICE6705 subfamily, lacking Tn4380 and having different VIM integrons located in another site of the elements. The remaining six non-ICE GIs represented miscellaneous structures. The presence of various integrons in the same ICE sublineage, and of the same integron in different GIs, indicated circulation and recombination of the integron-carrying genetic platforms across Pseudomonas species/genotypes. CONCLUSIONS: Despite the general diversity of the blaVIM-carrying GIs in Pseudomonas spp. in Poland, a clear predominance of broadly spread and rapidly evolving clc-type ICEs was documented, confirming their significant role in antimicrobial resistance epidemiology.

Asunto(s)

Islas Genómicas , Integrones , Infecciones por Pseudomonas , beta-Lactamasas , Polonia/epidemiología , beta-Lactamasas/genética , Integrones/genética , Humanos , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/epidemiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas/genética , Pseudomonas/enzimología , Pseudomonas/aislamiento & purificación , Antibacterianos/farmacología , Genotipo , Pruebas de Sensibilidad Microbiana , Elementos Transponibles de ADN/genética

RESUMEN

Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an SN2-like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Asunto(s)

Aminoácidos , Halogenación , Aminoácidos/metabolismo , Aminoácidos/química , Aminoácidos/biosíntesis , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/química , Arabidopsis/metabolismo , Arabidopsis/enzimología , Pseudomonas/metabolismo , Pseudomonas/enzimología , Ciclopropanos/química , Ciclopropanos/metabolismo

RESUMEN

The acyltransferase from Pseudomonas protegens (PpATase) catalyzes in nature the reversible transformation of monoacetylphloroglucinol to diacetylphloroglucinol and phloroglucinol. Interestingly, this enzyme has been shown to catalyze the promiscuous transformation of 3-hydroxyphenyl acetate to 2',4'-dihydroxyacetophenone, representing a biological version of the Fries rearrangement. In the present study, we report a mechanistic investigation of this activity of PpATase using quantum chemical calculations. A detailed mechanism is proposed, and the energy profile for the reaction is presented. The calculations show that the acylation of the enzyme is highly exothermic, while the acetyl transfer back to the substrate is only slightly exothermic. The deprotonation of the C6-H of the substrate is rate-limiting, and a remote aspartate residue (Asp137) is proposed to be the general base group in this step. Analysis of the binding energies of various acetyl acceptors shows that PpATase can promote both intramolecular and intermolecular Fries rearrangement towards diverse compounds.

Asunto(s)

Aciltransferasas , Pseudomonas , Pseudomonas/enzimología , Aciltransferasas/metabolismo , Aciltransferasas/química , Floroglucinol/química , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Termodinámica , Acilación , Modelos Moleculares , Biocatálisis , Especificidad por Sustrato

RESUMEN

Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle. The activity of these enzymes can be modulated by stimuli-sensing domains such as Per-ARNT-Sim (PAS). In Pseudomonas aeruginosa, more than 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual proteins possessing both canonical DGC and PDE motifs, that is, GGDEF and EAL, respectively. It was reported that deletion of the EAL/GGDEF dual enzyme PA0285, one of five c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms. PA0285 is anchored in the membrane and carries two PAS domains. Here, we confirm that its role is conserved in various P. aeruginosa strains and in Pseudomonas putida. Deletion of PA0285 impacts the early stage of colonization, and RNA-seq analysis suggests that expression of cupA fimbrial genes is involved. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds GTP and c-di-GMP, respectively, but only exhibits PDE activity in vitro. However, both GGDEF and EAL domains are important for PA0285 PDE activity in vivo. Complementation of the PA0285 mutant strain with a copy of the gene encoding the C-terminal GGDEF/EAL portion in trans was not as effective as complementation with the full-length gene. This suggests the N-terminal transmembrane and PAS domains influence the PDE activity in vivo, through modulating the protein conformation.

Asunto(s)

Proteínas Bacterianas , Pseudomonas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Pseudomonas/enzimología

RESUMEN

Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the δ-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases.

Asunto(s)

4-Hidroxibenzoato-3-Monooxigenasa , Pseudomonas , 4-Hidroxibenzoato-3-Monooxigenasa/metabolismo , Sitios de Unión , Flavoproteínas/genética , Flavoproteínas/metabolismo , Cinética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Filogenia , Pseudomonas/enzimología , Pseudomonas/metabolismo , Xylophilus/enzimología

RESUMEN

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Asunto(s)

Arabidopsis/crecimiento & desarrollo , Polifosfatos/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Microbiología del Suelo , Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/metabolismo , Arabidopsis/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Variación Genética , Ácidos Indolacéticos/metabolismo , Fósforo/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Filogenia , Pseudomonas/clasificación , Pseudomonas/enzimología , Rizosfera , Sideróforos/biosíntesis , Suelo/química

RESUMEN

Alginate lyases are epitomized as prospective therapeutic mediators for treating Pseudomonas aeruginosa infections, particularly in the cystic fibrosis airway through alginate degradation thereby improving the efficacy of anti-pseudomonal antibiotics. Investigation of metal-binding residues is significant for expounding the ion specificity of an enzyme and will provide a broad understanding of the potential roles of metal ions in enzyme function and stability. However, experimental analysis of metal ion-binding sites in proteins is time consuming and expensive. Concerning the clinical importance of this therapeutic enzyme, the present study was focused on the prediction and characterization of metal ion-binding sites of different alginate lyases reported in the literature through a computational approach using a Metal Ion-Binding Site Prediction and Docking Server. 3D structures of different alginate lyase from different organisms were retrieved, and these retrieved proteins were docked with twelve different metal ions such as Ca2+, Cu2+, Fe3+, Mg2+, Mn2+, Zn2+, Cd2+, Fe2+, Ni2+, Hg2+, Co2+, and Cu+. The binding affinity and interacting amino acids for alginate lyases produced by different microorganisms were compared and analysed. Further analysis on active site residues of reported alginate lyase and subsequent experiments will reveal the function of different metal ions in enhancing or inhibiting the catalysis of alginate lyase and will help in exploiting the enzyme as an efficient therapeutic agent as well as for industrial applications.

Asunto(s)

Proteínas Bacterianas/química , Metales/química , Simulación del Acoplamiento Molecular , Polisacárido Liasas/química , Pseudomonas/enzimología , Sphingomonas/enzimología , Sitios de Unión

RESUMEN

Our previous studies on FabG have identified two compounds 5-bromo-2-(thiophene-2-carboxamido) benzoic acid (A) and ethyl 6-bromo-2-((dimethylamino)methyl)-5-hydroxy-1-phenyl-1H-indole-3-carboxylate(B) as best hits with allosteric mode of inhibition. FabG is an integral part of bacterial fatty acid biosynthetic system FAS II shown to be an essential gene in most ESKAPE Pathogens. The current work is focussed on lead expansion of these two hit molecules which ended up with forty-three analogues (twenty-nine analogues from lead compound A and fourteen compounds from lead compound B). The enzyme inhibition studies revealed that compound 15 (effective against EcFabG, AbFabG, StFabG, MtFabG1) and 19 (inhibiting EcFabG and StFabG) had potency of broad-spectrum inhibition on FabG panel.

Asunto(s)

Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Pseudomonas/efectos de los fármacos , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Oxidorreductasas/metabolismo , Pseudomonas/enzimología , Relación Estructura-Actividad

RESUMEN

Tryptophan metabolism plays a role in the occurrence and development of hepatocellular carcinoma cells. By degrading certain amino acids, tumor growth can be limited while maintaining the body's normal nutritional requirements. Tryptophan side-chain oxidase (TSO) enzyme can degrade tryptophan, and its inhibitory effect on hepatocellular carcinoma cells is worthy of further study. To investigate the degradation effect on tryptophan, TSO was isolated and purified from qq Pseudomonas. The reaction products were identified with high performance liquid chromatography (HPLC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS). De novo sequencing provided the complete amino acid sequence of TSO. The results of CCK-8, colony formation, transwell, and qPCR confirmed that TSO had inhibitory effects on the proliferation and migration of HCCLM3 (human hepatocarcinoma cell line) and HepG2 cells. The results of flow cytometry confirmed its apoptotic activity. In animal experiments, we found that the tumor-suppressive effect was better in the oncotherapy group than the intraperitoneal injection group. The results of immunohistochemistry also suggested that TSO could inhibit proliferation and promote apoptosis. In conclusion, a specific enzyme that can degrade tryptophan and inhibit the growth of hepatoma cells was authenticated, and its basic information was obtained by extraction/purification and amino acid sequencing.

Asunto(s)

Antineoplásicos/farmacología , Proteínas Bacterianas/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Oxigenasas de Función Mixta/farmacología , Triptófano/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Oxigenasas de Función Mixta/biosíntesis , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/aislamiento & purificación , Modelos Moleculares , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Estructura Secundaria de Proteína , Pseudomonas/química , Pseudomonas/enzimología , Pseudomonas/genética , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

Nonribosomal peptide synthetases (NRPSs) are typically multimodular enzymes that assemble amino acids or carboxylic acids into complex natural products. Here, we characterize a monomodular NRPS, PvfC, encoded by the Pseudomonas virulence factor (pvf) gene cluster that is essential for virulence and signaling in different bacterial species. PvfC exhibits a unique adenylation-thiolation-reductase (ATR) domain architecture that is understudied in bacteria. We show that the activity of PvfC is essential in the production of seven leucine-derived heterocyclic natural products, including two pyrazines, a pyrazinone, and a rare disubstituted imidazole, as well as three pyrazine N-oxides that require an additional N-oxygenation step. Mechanistic studies reveal that PvfC, without a canonical peptide-forming domain, makes a dipeptide aldehyde intermediate en route to both the pyrazinone and imidazole. Our work identifies a novel biosynthetic route for the production of pyrazinones, an emerging class of signaling molecules and virulence factors. Our discovery also showcases the ability of monomodular NRPSs to generate amino acid- and dipeptide-aldehydes that lead to diverse natural products. The diversity-prone biosynthesis by the pvf-encoded enzymes sets the stage for further understanding the functions of pvf in bacterial cell-to-cell signaling.

Asunto(s)

Productos Biológicos/química , Péptido Sintasas/metabolismo , Factores de Virulencia/química , Aldehídos/química , Aminoácidos/química , Ácidos Carboxílicos/química , Óxidos N-Cíclicos/química , Dipéptidos/química , Imidazoles/química , Pseudomonas/enzimología , Pirazinas/química

RESUMEN

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.

Asunto(s)

Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/genética , Formiato Deshidrogenasas/genética , NADPH-Ferrihemoproteína Reductasa/genética , Ingeniería de Proteínas/métodos , Bacillus megaterium/enzimología , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas Bacterianas/metabolismo , Catálisis , Sistema Enzimático del Citocromo P-450/metabolismo , Enzimas/genética , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidación-Reducción , Pseudomonas/enzimología , Pseudomonas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología