The dual GGDEF/EAL domain enzyme PA0285 is a Pseudomonas species housekeeping phosphodiesterase regulating early attachment and biofilm architecture.

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.

Protocatechuate hydroxylase is a novel group A flavoprotein monooxygenase with a unique substrate recognition mechanism.

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.

Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

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.

Scrutiny of Metal Ion Binding Sites in Different Alginate Lyases through In Silico Analysis.

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.

Lead derivatization of ethyl 6-bromo-2-((dimethylamino)methyl)-5-hydroxy-1-phenyl-1H-indole-3-carboxylate and 5-bromo-2-(thiophene-2-carboxamido) benzoic acid as FabG inhibitors targeting ESKAPE pathogens.

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.

Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity.

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.

Enzymatic Synthesis of Diverse Heterocycles by a Noncanonical Nonribosomal Peptide Synthetase.

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.

Tryptophan Side-Chain Oxidase Enzyme Suppresses Hepatocellular Carcinoma Growth through Degradation of Tryptophan.

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.

Magnetic ZIF-8-Based Mimic Multi-enzyme System as a Colorimetric Biosensor for Detection of Aryloxyphenoxypropionate Herbicides.

In the present study, a magnetic mimic multi-enzyme system was developed by encapsulating the aryloxyphenoxypropionate (AOPP) herbicide hydrolase QpeH and alcohol oxidase (AOx) in zeolitic imidazolate framework (ZIF-8) nanocrystals with magnetic Fe3O4 nanoparticles (MNPs) to detect AOPP herbicides. The structural, protein loading capacity and loading ratio, porosity, and magnetic properties of QpeH/AOx@mZIF-8 were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, nitrogen sorption, and vibrating sample magnetometry. An AOPP herbicide colorimetric biosensor made with QpeH/AOx@mZIF-8 had the highest sensitivity toward quizalofop-P-ethyl (QpE) with a limit of detection of 8.2 µM. This system was suitable to detect two other AOPP herbicides, including fenoxaprop-P-ethyl (FpE) and haloxyfop-P-methyl (HpE). The practical application of the biosensor was verified through quantitative analysis of QpE residues in industrial wastewater and field soils. Furthermore, QpeH/AOx@mZIF-8 exhibited excellent long-term storage stability (at least 50 days), easy separation by magnet, and reusability (at least 10 cycles), supporting its promising role in simple and low-cost detection of AOPP herbicides in real environmental samples.

Computational Elucidation of Phylogenetic, Functional and Structural Features of Methioninase from Pseudomonas, Escherichia, Clostridium and Citrobacter Strains.

BACKGROUND: L-Methioninase (EC 4.4.1.11; MGL) is a pyridoxal phosphate (PLP)-dependent enzyme that is produced by a variety of bacteria, fungi, and plants. L-methioninase, especially from Pseudomonas and Citrobacter sp., is considered as the efficient therapeutic enzyme, particularly in cancers such as glioblastomas, medulloblastoma, and neuroblastoma that are more sensitive to methionine starvation. OBJECTIVE: The low stability is one of the main drawbacks of the enzyme; in this regard, in the current study, different features of the enzyme, including phylogenetic, functional, and structural from Pseudomonas, Escherichia, Clostridium, and Citrobacter strains were evaluated to find the best bacterial L-Methioninase. METHODS: After the initial screening of L-Methioninase sequences from the above-mentioned bacterial strains, the three-dimensional structures of enzymes from Escherichia fergusonii, Pseudomonas fluorescens, and Clostridium homopropionicum were determined through homology modeling via GalaxyTBM server and refined by GalaxyRefine server. RESULTS AND CONCLUSION: Afterwards, PROCHECK, verify 3D, and ERRAT servers were used for verification of the obtained models. Moreover, antigenicity, allergenicity, and physico-chemical analysis of enzymes were also carried out. In order to get insight into the interaction of the enzyme with other proteins, the STRING server was used. The secondary structure of the enzyme is mainly composed of random coils and alpha-helices. However, these outcomes should further be validated by wet-lab investigations.

Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase.

Two recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ, allowed to successfully produce and secrete recombinant αEstS9Δ enzyme outside of the host cell. The purified αEstS9Δ esterase was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the acetate (C2) ester. The single-domain αEstS9Δ esterase exhibits the highest activity at 60oC and pH 9.5. In addition, the enzyme retains 90% of its activity after 3 hour incubation at 70-90oC. What should be also noted is that αEstS9Δ esterase produced in the K. phaffii expression system has a much higher specific activity (0.069 U/mg of protein) than the recombinant EstS9Δ esterase produced in an E. coli expression system (0.0025 U/mg of protein) (Wicka et al., 2016, Acta Biochim Pol 63: 117-125. https://doi.org/10.18388/abp.2015_1074).

Screening, Cloning, Expression and Characterization of New Alkaline Trehalose Synthase from Pseudomonas monteilii and Its Application for Trehalose Production.

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli. Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40°C, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

Characterization of a Stable Form of Carboxypeptidase G2 (Glucarpidase), a Potential Biobetter Variant, From Acinetobacter sp. 263903-1.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme widely used to detoxify methotrexate (MTX) and in enzyme/prodrug therapy for cancer treatment. However, several drawbacks, such as instability, have limited its efficiency. Herein, we have evaluated the properties of a putative CPG2 from Acinetobacter sp. 263903-1 (AcCPG2). AcCPG2 is compared with a CPG2 derived from Pseudomonas sp. strain RS-16 (PsCPG2), available as an FDA-approved medication called glucarpidase. After modeling AcCPG2 using the I-TASSER program, the refined model was validated by PROCHECK, VERIFY 3D and according to the Z score of the model. Using computational analyses, AcCPG2 displayed higher thermodynamic stability and a lower aggregation propensity than PsCPG2. AcCPG2 showed an optimum pH of 7.5 against MTX and was stable over a pH range of 5-10. AcCPG2 exhibited optimum activity at 50 °C and higher thermal stability at a temperature range of 20-70 °C compared to PsCPG2. The Km value of the purified AcCPG2 toward folate and MTX was 31.36 µM and 44.99 µM, respectively. The Vmax value of AcCPG2 for folate and MTX was 125.80 µmol/min/mg and 48.90  µmol/min/mg, respectively. Accordingly, thermostability and pH versatility makes AcCPG2 a potential biobetter variant for therapeutic applications.

Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from Pseudomonas species.

L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.

Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation.

BACKGROUND: Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. RESULTS: Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. CONCLUSIONS: The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.

Physiological Role of the Previously Unexplained Benzenetriol Dioxygenase Homolog in the Burkholderia sp. Strain SJ98 4-Nitrophenol Catabolism Pathway.

4-Nitrophenol, a priority pollutant, is degraded by Gram-positive and Gram-negative bacteria via 1,2,4-benzenetriol (BT) and hydroquinone (HQ), respectively. All enzymes involved in the two pathways have been functionally identified. So far, all Gram-negative 4-nitrophenol utilizers are from the genera Pseudomonas and Burkholderia. But it remains a mystery why pnpG, an apparently superfluous BT 1,2-dioxygenase-encoding gene, always coexists in the catabolic cluster (pnpABCDEF) encoding 4-nitrophenol degradation via HQ. Here, the physiological role of pnpG in Burkholderia sp. strain SJ98 was investigated. Deletion and complementation experiments established that pnpG is essential for strain SJ98 growing on 4-nitrocatechol rather than 4-nitrophenol. During 4-nitrophenol degradation by strain SJ98 and its two variants (pnpG deletion and complementation strains), 1,4-benzoquinone and HQ were detected, but neither 4-nitrocatechol nor BT was observed. When the above-mentioned three strains (the wild type and complementation strains with 2,2'-dipyridyl) were incubated with 4-nitrocatechol, BT was the only intermediate detected. The results established the physiological role of pnpG that encodes BT degradation in vivo. Biotransformation analyses showed that the pnpA-deleted strain was unable to degrade both 4-nitrophenol and 4-nitrocatechol. Thus, the previously characterized 4-nitrophenol monooxygenase PnpASJ98 is also essential for the conversion of 4-nitrocatechol to BT. Among 775 available complete genomes for Pseudomonas and Burkholderia, as many as 89 genomes were found to contain the putative pnpBCDEFG genes. The paucity of pnpA (3 in 775 genomes) implies that the extension of BT and HQ pathways enabling the degradation of 4-nitrophenol and 4-nitrocatechol is rarer, more recent, and likely due to the release of xenobiotic nitroaromatic compounds. IMPORTANCE An apparently superfluous gene (pnpG) encoding BT 1,2-dioxygenase is always found in the catabolic clusters involved in 4-nitrophenol degradation via HQ by Gram-negative bacteria. Our experiments reveal that pnpG is not essential for 4-nitrophenol degradation in Burkholderia sp. strain SJ98 but instead enables its degradation of 4-nitrocatechol via BT. The presence of pnpG genes broadens the range of growth substrates to include 4-nitrocatechol or BT, intermediates from the microbial degradation of many aromatic compounds in natural ecosystems. In addition, the existence of pnpCDEFG in 11.6% of the above-mentioned two genera suggests that the ability to degrade BT and HQ simultaneously is ancient. The extension of BT and HQ pathways including 4-nitrophenol degradation seems to be an adaptive evolution for responding to synthetic nitroaromatic compounds entering the environment since the industrial revolution.

Atrazine detoxification by intracellular crude enzyme extracts derived from epiphytic root bacteria associated with emergent hydrophytes.

The present study demonstrated atrazine detoxification by intracellular crude enzyme extracts of Pseudomonas spp. strains ACB and TLB. Indigenous bacterial protein-based remediation techniques could be an alternative to bioaugmentation which pose multiple challenges when applied to the field. Intracellular enzymes were extracted from strains ACB and TLB and their degradation potential of 10 mg L-1 was determined using Gas Chromatography; further, enzyme extracts were subjected to protein profiling studies. In span of 6 h, enzyme extracts of strain ACB showed maximum degradation at 30 °C and 40 °C (71%) and enzyme extracts of strain TLB showed maximum degradation at 40 °C (48%). Atrazine degradation by enzyme extracts of strain ACB showed maximum degradation at pH 7 (71%) and pH 6 (69%) in 6 h. Similarly, enzyme extracts of strain TLB showed maximal degradation at pH 6 (46%) in 6 h. The present study demonstrated, for the first time, efficient atrazine remediation by intracellular crude enzyme extracts from epiphytic root bacteria at a range of temperature and pH conditions. Protein profiling studies indicated that atrazine induced expression of CoA ester lyase and alkyl hydroperoxide reductase in the strains ACB and TLB respectively. Expressions of these proteins have never been associated with atrazine exposure.

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αß)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis.

Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation.

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Furthermore, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, that is, isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for KS (9.3-fold higher) and kcat (4.8-fold lower) compared with corresponding KM and kcat values of isolated CHMO, but a favorable KI for cyclohexanone (5.3-fold higher). The unfavorable KS and kcat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, to exploit the high potential of biofilms regarding process stability. Suspended cells showed only 1.8-fold higher KS , but 1.3- and 4.2-fold higher kcat and KI values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2.

Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The Km and kcat values of 6.5 µM and 4.57 s-1, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.