Purification and Properties of 2-Halo Acid Dehalogenase from Pseudomonas putida

Previous studies of potato varieties indicated that changes during cooking could be mathematically described and that some chemical components and the cell size may influence the cooking behavior. To find out whether the same principles can be adopted for other root vegetables, the cooking behavior of three other low-starch root vegetables were investigated and the results compared. Slices (6 mm thick and 30 mm diameter) were treated in water at 100°C. Mathematical expressions were assessed, and coefficients were determined to describe the kinetic behavior of the products. The cell size and pectin content of the raw materials determined the cooking characteristics. Texture development could be predicted by shear force measurements.     

Purification and characterization of trans-3-(4-methoxyphenyl) glycidic acid methyl ester hydrolyzing lipase from Pseudomonas aeruginosa

Enantiospecific lipase was purified from Pseudomonas aeruginosa MTCC 5113 and it was used for the hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate, a key intermediate in the synthesis of cardiovascular drug, diltiazem. Enzyme from broth supernatant was precipitated with acetone and purified by anion exchange and gel filtration chromatography. The purified lipase was a homogenous protein having a molecular weight of 59.4 kDa as determined by SDS-PAGE. Isoelectric point was found to be approximately 5.5 after 2D electrophoresis. This organic solvent tolerant enzyme was found to be active in presence of EDTA, Tween-80 and β-mercaptoethanol whereas sodium dodecyl sulphate and dithiothreitol inhibited its activity. The K_m and V_max of the enzyme were 50 mM and 27.1 μmol/min mg, respectively using p-nitrophenyl palmitate as a substrate. The activity of lipase was confirmed by (±)-MPGM hydrolysis and zymography.     

Characterization of 3-Guanidinopropionate Amidinohydrolase from Pseudomonas aeruginosa and a Comparative Study with 4-Guanidinobutyrate Amidinohydrolase from another Pseudomonas

3-Guanidinopropionate amidinohydrolase, a new enzyme (EC class 3.5.3), was purified 220-fold from Pseudomonas aeruginosa PAO 1 grown on 3-guanidinopropionate. The enzyme was found to be essentially homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was estimated to be 195,000-215,000. The subunit molecular weight was estimated to be 36,000. The optimal pH was 9.0. The Km value for 3-guanidinopropionate was 45 mm. Incubation of the enzyme with EDTA in potassium phosphate buffer, pH 7.0, at 40°C resulted in almost complete inactivation, and the inactive enzyme was specifically reactivated by Mn²⁺. Taurocyamine (11%) and 4-guanidinobutyrate (3%) were hydrolyzed as fast as 3-guanidinopropionate at the relative rates indicated. The enzyme was inactivated by p-chloromercuribenzoic acid and the inactive enzyme was reactivated by incubation with 2-mercaptoethanol. Coelectrophoresis of the enzyme with 4-guanidinobutyrate amidinohydrolase purified from Pseudomonas sp. ATCC 14676 in polyacrylamide gels in the presence and absence of sodium dodecyl sulfate demonstrated their close mobilities. 4-Aminobutyrate, propionate, and n-butyrate were common competitive inhibitors of these enzymes. The evolutionary relationship between the two enzymes was discussed.     

Structural and Catalytic Properties of the D-3-Hydroxybutyrate Dehydrogenase from Pseudomonas aeruginosa

To put forward BDH from Pseudomonas aeruginosa’s enzymatic properties, we report a two-step purification of BDH and its gene sequencing allowing the investigation of its structural properties. Purification of BDH was achieved, using ammonium sulfate fractionation and Blue Sepharose CL-6B affinity chromatography. SDS–PAGE analysis reveals a MM of 29 kDa, whereas the native enzyme showed a MM of 120 kDa suggesting a homotetrameric structure. BDH encoding gene sequence shows a nucleotide open reading frame sequence of 771 bp encoding a 265 amino acid residues polypeptide chain. The modeling analysis of the three dimensional structure fits with the importance of amino acids in the catalysis reaction especially a strictly conserved tetrad. Amino-acid residues in interaction with the coenzyme NAD⁺ were also identified.     

Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.     

Mechanistic and Structural Origins of the Asymmetric Barrier to Prion-like Cross-Seeding between Tau-3R and Tau-4R

The spread and deposition of infectious fibrillar protein aggregates in the brain via a prion-like mechanism is a critical component in the patho-physiology of various neurodegenerative diseases, including the tauopathies. In tauopathies, two isoforms of tau, containing three and four microtubule binding repeats, are found to aggregate, and the type of isoform present in aggregates determines the type of tauopathy. Cross-seeding between the two tau isoforms is limited by an asymmetric barrier similar to the species barrier that restricts prion transmission across species, whose origin has remained unclear. In this study, the growth of the tau fibrils is shown to be describable by a two-step Michaelis–Menten-like model. Delineation of the mechanism as a Michaelis–Menten-like mechanism has enabled a quantitative understanding of the asymmetric seeding barrier that exists between two isoforms of tau, tau-K18 and tau-K19 (which differ in containing four and three microtubule binding repeats, respectively), wherein tau-K18 fibrils cannot seed tau-K19 monomer. Furthermore, high-resolution structural analysis of the two isoforms shows that the structural core is more ordered in tau-K19 than in tau-K18. Hence, the current work provides kinetic and structural rationales for asymmetric seeding barriers in general and for the two tau isoforms in particular.     

PK-sensitive PrPSc Is Infectious and Shares Basic Structural Features with PK-resistant PrPSc

One of the main characteristics of the transmissible isoform of the prion protein (PrP^Sc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP^Sc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP^Sc that is sensitive to PK hydrolysis (sPrP^Sc). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP^Sc versus the PK-resistant (rPrP^Sc) fractions of PrP^Sc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP^Sc and rPrP^Sc fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP^Sc to the rPrP^Sc forms of PrP^Sc. Although the sPrP^Sc and rPrP^Sc fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.     

Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from Pseudomonas putida

4-Hydroxyphenylacetic acid 3-hydroxylase is a key enzyme in the pathway for the microbial degradation of phenylalanine, tyrosine and many aromatic amines. This enzyme was purified to homogeneity from Pseudomonas putida by affinity chromatography. The protein had a molecular weight of 91,000 and was a dimer of identical subunits. It was a typical external flavoprotein monooxygenase and showed an absolute requirement of NADH for activity. The enzyme had a pH optimum of 7.5 and the Km values for 4-hydroxyphenylacetic acid and NADH were 2 x 10(-4) M and 5.9 x 10(-5) M respectively. It was strongly inhibited by heavy metal ions and thiol reagents, suggesting the possible involvement of -SH group(s) in enzyme reaction.     

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.     

Isolation and Identification of trans-2- and trans-3-Hydroxy-1,8-cineole Glucosides from Alpinia galanga

Three hydroxy-1,8-cineole glucopyranosides, (1R,2R,4S)- and (1S,2S,4R)-trans-2-hydroxy-1,8-cineole β-D-glucopyranosides, and (1R,3S,4S)-trans-3-hydroxy-1,8-cineole β-D-glucopyranoside, which are possible precursors of acetoxy-1,8-cineoles as unique aroma components, were isolated from the rhizomes of greater galangal (Alpinia galanga W.). Their structures were analyzed by FAB-MS and NMR spectrometry, and the absolute configulation of each aglycone was determined by using a GC-MS analysis with a capillary column coated with a chiral stationary phase. The composition of the diastereomers of (1R,2R,4S)- and (1S,2S,4R)- trans-2-hydroxy-1,8-cineole β-D-glucopyranosides in the rhizomes was determined as 3:7 by a GC-MS analysis after preparing the trifluoroacetate derivatives of the glucosides.     

Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil

Pseudomonas aeruginosa strain SP4, isolated from petroleum-contaminated soil in Thailand, was used to produce a biosurfactant from a nutrient broth with palm oil as the carbon source. The key components of the crude biosurfactant were fractionated by using HPLC-ELSD technique. With the use of ATR-FTIR spectroscopy, in combination with (1)H NMR and MS analyses, chemical structures of the fractionated components of the crude biosurfactant were identified as rhamnolipid species. When compared to synthetic surfactants, including Pluronic F-68, which is a triblock nonionic surfactant containing poly(ethylene oxide) and poly(propylene oxide), and sodium dodecyl sulfate, the crude biosurfactant showed comparable physicochemical properties, in terms of the surface activities. The crude biosurfactant reduced the surface tension of pure water to 29.0 mN/m with a critical micelle concentration of approximately 200 mg/l, and it exhibited good thermal and pH stability. The crude biosurfactant also formed stable water-in-oil microemulsions with crude oil and various types of vegetable oils, but not with short-chain hydrocarbons.     

Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the Free Fatty Acid Receptor FFA4

In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr³⁴⁷, Thr³⁴⁹, Ser³⁵⁰, Ser³⁵⁷, and Ser³⁶⁰) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu³⁴¹, Asp³⁴⁸, and Asp³⁵⁵ located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from G_q/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling.     

Mechanistic information on the reaction of cis- and trans-[RuCl2(DMSO)4] with d(T2GGT2) derived from MALDI-TOF and HPLC studies

Reactions of trans and cis isomers of the Ru(II) complex [RuCl(2)(DMSO)(4)] with single-stranded hexanucleotide d(T(2)GGT(2)) were studied in aqueous solutions in the absence and presence of excess chloride by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Despite the different reactive species formed from the two isomers in aqueous solution, similar reaction products are obtained in their interaction with d(T(2)GGT(2)). Both [RuCl(2)(DMSO)(4)] isomers bind to the oligonucleotide in the bidentate mode to form thermodynamically stable bis-guanosine adducts, Ru(G-N7)(2). Significant differences were observed in the reaction rates, however the reaction with trans- [RuCl(2)(DMSO)(4)] is ca. 5-10 times faster in comparison to that observed for the cis analogue. This difference is interpreted in terms of different rate-limiting steps for the trans and cis complexes, respectively. It is suggested that the rate of the reaction with the trans isomer is controlled by dissociation of a Cl(-) ligand from the initially formed trans,cis,cis-[RuCl(2)(DMSO)(2)(H(2)O)(2)]. In the contrast, release of a dimethyl sulfoxide molecule from the reactive species cis,fac-[RuCl(2)(DMSO)(3)(H(2)O)] is likely to be rate limiting for the cis analogue. Significant influence of electrostatic interactions on the reaction rate was observed for the trans isomer. Mechanistic interpretation of the observed reactivity trends based on data obtained from UV-Vis spectroscopy, HPLC and MALDI-TOF MS studies is presented and discussed within the paper.     

Purification and characterization of the 4-hydroxyphenylacetic acid-3-hydroxylase from Pseudomonas putida U

4-Hydroxyphenylacetic acid-3-hydroxylase from Pseudomonas putida U was purified to homogeneity (96-fold) from bacterial cultures grown in a chemically defined medium containing 4-hydroxyphenylacetic acid as the sole carbon source. The maximal rate of catalysis occurred at pH 7.5 and 40°C. Under these conditions, the K_m values calculated for 4-hydroxyphenylacetic acid, NADH and FAD were 38, 41 and 4 μM respectively. The native enzyme (M_r 65 000) had two identical subunits in an α₂ oligomeric structure and required the addition of FAD, so it was classified as an external flavoprotein monooxygenase. 4-Hydroxyphenylacetic acid-3-hydroxylase showed a broad substrate range. It was specifically induced by 4-hydroxyphenylacetic acid, although phenylacetic acid and some phenyl-alkanoic acids also induced enzymatic activity to a lesser extent. 4-Hydroxyphenylacetic acid-3-hydroxylase induction and 4-hydroxyphenylacetic acid consumption were unaffected by the presence of glucose, suggesting that the uptake and hydroxylation of 4-hydroxyphenylacetic acid are not under carbon catabolite repression.     

Purification of Stearidonic Acid (18:4(n-3)) and Hexadecatetraenoic Acid (16:4(n-3)) from Algal Fatty Acid with Lipase and Medium Pressure Liquid Chromatography

Stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) are included in some edible marine algae such as Undaria pinnatifida and Ulva pertusa with relatively high compositions (up to 40%) of total fatty acids. In order to prepare 16:4(n-3) and 18:4(n-3) enriched fatty acid concentrates, we screened for a suitable lipase which concentrates these acids by the removal of other fatty acids in the selective esterification reaction reported by Shimada et al. (Shimada et al. (1997), J. Am. Oil Chem. Soc., 74, 1465-1470). In combination with the lipase reaction and reversed-phase medium pressure liquid chromatography, we purified 18:4(n-3) and 16:4(n-3) to more than 95% purity.     

Protomitohondria from liver cells: Similarities with and differences from mitochondria

In this study, we continue the investigation of a number of the properties of protomitochondria, which are young mitochondria of a smaller size that are present in animal cells. The protomitochondria were obtained from the rat liver by filtration of a total mitochondrial suspension through Millipore filters. The organelles contain the active respiratory chain as shown by the high rate of oxygen consumption during succinate and NADH oxidation. The shunt activity of succinate-tetrazolium reductase was lower and the activity of NADH-tetrazolium reductase was higher as compared with those in mitochondria. According to gel electrophoresis and gel filtration no qualitative differences between protomitochondria (0.25–0.45 µm) and mitochondria in their major protein composition were found. Nevertheless, there were some quantitative differences in several bands. Perhaps these differences reflect the process of intracellular maturation of protomitochondria to mitochondria. The data we obtained are important for understanding mitochondriogenesis in the animal cell.     

Identification of the Biosynthetic Gene Cluster for the Pseudomonas aeruginosa Antimetabolite l-2-Amino-4-Methoxy-trans-3-Butenoic Acid

l-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections and is considered the main pathogen responsible for chronic pneumonia in cystic fibrosis patients (7, 23). P. aeruginosa also infects other organisms, such as insects (4), nematodes (6), plants (18), and amoebae (20). Its ability to thrive as a pathogen and to compete in aquatic and soil environments can be partly attributed to the production and interplay of secreted virulence factors and secondary metabolites. While the importance of many of these exoproducts has been studied, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid (AMB; methoxyvinylglycine) (Fig. ​(Fig.1)1) has received only limited attention. Identified during a search for new antibiotics, AMB was found to reversibly inhibit the growth of Bacillus spp. (26) and Escherichia coli (25) and was later shown to inhibit the growth and metabolism of cultured Walker carcinosarcoma cells (28). AMB is a γ-substituted vinylglycine, a naturally occurring amino acid with a β,γ-C=C double bond. Other members of this family are aminoethoxyvinylglycine from Streptomyces spp. (19) and rhizobitoxine, made by Bradyrhizobium japonicum (16) and Pseudomonas andropogonis (15) (Fig. ​(Fig.1).1). As inhibitors of pyridoxal phosphate-dependent enzymes (13, 17, 21, 22), γ-substituted vinylglycines have multiple targets in bacteria, animals, and plants (3, 5, 10, 21, 22, 29). However, the importance of AMB as a toxin in biological interactions with P. aeruginosa has not been addressed, as AMB biosynthesis and the genes involved have not been elucidated.Open in a separate windowFIG. 1.Chemical structures of the γ-substituted vinylglycines AMB, aminoethoxyvinylglycine, and rhizobitoxine.     

Production of a Novel Compound, 7,10,12-Trihydroxy-8(E)-Octadecenoic Acid from Ricinoleic Acid by Pseudomonas aeruginosa PR3

The production and its potential use of a novel trihydroxy unsaturated fatty acid, 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD), were investigated. TOD was formed by Pseudomonas aeruginosa PR3 (NRRL B-18602) in a culture supplied with exogenous ricinoleic acid. The yield of TOD production was always higher in a rich culture medium than in minimal screening medium. Extending the conversion time from 48 to 72 h prior to lipid extraction led to a 65% reduction in yield, indicating that TOD was further metabolized by strain PR3 and that control of reaction time is important to achieving a maximum yield. The optimum culture density, reaction time, pH, temperature, and substrate concentration for the production of TOD were: 20–24 h culture growth, 48 h, 7.0, 25°C, and 1% (vol/vol), respectively. Under optimum conditions, the yield of TOD production was greater than 45%. TOD was found to be an antifungal agent most active against the fungus that causes blast disease in rice plants, the most important fungal disease affecting rice production worldwide. Received: 4 January 2001/Accepted: 6 February 2001