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At least 433 records · Page 24Linked to original sources
PubMed1999

Mechanistic insights from the three-dimensional structure of 3-oxo-Delta(5)-steroid isomerase.

3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through the formation of an intermediate dienolate. The three-dimensional structure of the enzyme from Pseudomonas testosteroni was solved by multidimensional heteronuclear magnetic resonance spectroscopy. This protein, a 28-kDa symmetric dimer, exhibits a three-dimensional fold with the two independently folded monomers packed together via extensive hydrophobic and electrostatic interactions. The previously identified catalytically important residues Tyr-14 (general acid) and Asp-38 (general base) are located near the bottom of a deep hydrophobic cavity and are positioned in a manner consistent with previous mechanistic hypotheses. The structure also revealed the presence of an unexpected acid group (Asp-99) located in the active site adjacent to Tyr-14. Mutagenesis and kinetic studies show that Asp-99 has an anomalously high pK(a) (>9), which allows it to contribute to catalysis by donating a hydrogen bond to the intermediate and to the transition states. In support of this hypothesis, effects on the kinetic parameters of the mutations Y14F and D99A are additive in the Y14F/D99A mutant.

Comamonas testosteroni↗
PubMed2006

Degradation of fumonisin B1 by a bacterial strain isolated from soil.

A mixed microbial culture degrading fumonisin B l was obtained from soil samples using an enrichment culture procedure. A bacterial isolate from the enrichment culture (strain NCB 1492) degraded fumonisin B1 after incubation for 3 h, as indicated by TLC and HPLC analysis. On the basis of the sequence analysis of 16S rDNA, strain NCB 1492 was related to the Delftia/Comamonas group. Thin-layer chromatographic analysis indicated the presence of metabolites in the NCB 1492 culture filtrates after degradation of fumonisin B1 supplied as sole carbon and nitrogen source in phosphate buffer. Four metabolites were identified by mass spectrometry analysis.

Biodegradation, Environmental↗
PubMed2005

An enzymatic cycling method for the determination of serum total bile acids with recombinant 3alpha-hydroxysteroid dehydrogenase.

A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay. We constructed a prokaryotic expression system to prepare the recombinant 3alpha-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay. The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm. The enzymatic cycling method could detect 0.22 micromol/L total bile acids in serum. Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively. The calibration curve for total bile acids in serum was linear between 0.5 and 180 micromol/L. This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase. In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3alpha-hydroxysteroid dehydrogenase as the tool enzyme.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗
PubMed2004

Analysis of C-3 epimerization in (24R)-24,25-dihydroxyvitamin D3 catalyzed by hydroxysteroid dehydrogenase.

Studies on the C-3 epimerization in (24R)-24,25-dihydroxyvitamin D(3) [24R,25(OH)(2)D(3)] were performed using hydroxysteroid dehydrogenases (HSDs). 3-Epi-24R,25(OH)(2)D(3) was formed from 24R,25(OH)(2)D(3) by the catalysis of 3alpha- or beta-HSD. These HSDs also catalyzed the C-3 epimerization in 3-epi-24R,25(OH)(2)D(3) to form 24R,25(OH)(2)D(3). 24R,25(OH)(2)D(3) and its C-3 epimer were separated by inclusion high-performance liquid chromatography using gamma-cyclodextrin (gamma-CD) as the mobile phase additive or a gamma-CD bonded chiral column. The product derived from the intermediate during the C-3 epimerization was isolated from the incubation specimens and identified as (7Z)-(24R)-24,25-dihydroxy-9,10-secocholesta-4,7,10(19)-trien-3-one by several instrumental analyses including (1)H-nuclear magnetic resonance spectrometry. The occurrence of this compound strongly proves that the formation of the C-3 epimer by HSD involves a dehydrogenation process. The present study suggests that HSDs may catalyze the C-3 epimerization of vitamin D compounds and modulate their concentrations and biological activities in animals and humans.

24,25-Dihydroxyvitamin D 3↗
PubMed2001

Utility of high performance liquid chromatography/electrospray/mass spectrometry of polar lipids in specifically Per-13C labeled Gram-negative bacteria DA001 as a tracer for acceleration of bioremediation in the subsurface.

Specific fatty acids from phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) recovered from a per 13C-labeled bacteria can be detected in environmental samples and used as measures of bacterial transport in the subsurface. Detection of palmitic acid (16:0) and oleic acid (18:1) at m/z 271 (255+16) and 299 (281+18) as negative ions in PG and PE separated by high performance liquid chromatography (HPLC) and detected after up-front collisionally induced dissociation (CID) utilizing electrospray (ES) mass spectrometry (MS) provided sufficient sensitivity and specificity for detection in the presence of the indigenous microbiota. Application of tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) was use to monitor selected transitions. MRM can increase the sensitivity so that polar lipids recovered from cell densities currently at about 10(4) cells/sample can be detected. This technology provides a non-intrusive mechanism for monitoring the distribution of bacteria added to accelerate in situ bioremediation of subsurface sediments.

Biodegradation, Environmental↗
PubMed2003

Tracking of injected and resident (previously injected) bacterial cells in groundwater using ferrographic capture.

A high-resolution bacterial tracking technique, ferrographic capture, was used to enumerate fluorescent-stained bacterial cells that were injected into groundwater during a field experiment. The goal of the experiment was to investigate whether detachment of previously injected stained resident cells attached to aquifer sediment was enhanced in the presence of the newly injected mobile cells. This injection was an improvement on past experiments in that the attached (resident) cells were stained, allowing their concentrations to be enumerated directly by ferrographic capture (upon detachment). Contrary to expectations based on previous experiments, enhanced detachment of stained resident cells did not occur upon the arrival of injected cells. Consistent with previous experiments, however, was the observation of ephemeral increases in unstained cell concentrations coincident with the arrival of the stained injected cells. The ephemeral pulses of unstained cells were previously speculated to represent enhanced detachment of unstained indigenous cells in response to hydrodynamic collision with injected cells. The lack of enhanced detachment of stained resident cells in the present experiments indicates that increased concentrations of unstained cells may have occurred by mechanisms other than hydrodynamic collision. Visually observed variations in stain intensity indicated that increased unstained cell concentrations may have resulted from cell division at the low-concentration fringe of the injected plume.

Aerobiosis↗
PubMed2000

Role of catalytic residues in enzymatic mechanisms of homologous ketosteroid isomerases.

Ketosteroid isomerase (KSI) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a diffusion-controlled rate. In this study of KSI, we have detailed the structures of its active site, the role of various catalytic residues, and have explained the origin of the its fast reactivity by carrying out a detailed investigation of the enzymatic reaction mechanism. This investigation included the X-ray determination of 15 crystal structures of two homologous enzymes in free and complexed states (with inhibitors) and extensive ab initio calculations of the interactions between the active sites and the reaction intermediates. The catalytic residues, through short strong hydrogen bonds, play the role of charge buffer to stabilize the negative charge built up on the intermediates in the course of the reaction. The hydrogen bond distances in the intermediate analogues are found to be about 0.2 A shorter in the product analogues both experimentally and theoretically.

Aspartic Acid↗
PubMed2001

15N NMR relaxation studies of backbone dynamics in free and steroid-bound Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni.

The backbone dynamics of Delta(5)-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been studied in free enzyme and its complex with a steroid ligand, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S(2), tau(e), and R(ex)) and the overall rotational correlation time (tau(m)). The rotational correlation times were 19.23 +/- 0.08 and 17.08 +/- 0.07 ns with the diffusion anisotropies (D( parallel)/D( perpendicular)) of 1.26 +/- 0.03 and 1.25 +/- 0.03 for the free and steroid-bound KSI, respectively. The binding of 19-NTHS to free KSI causes a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in helix A1 and strand B4. However, the majority of the residues exhibit reduced order parameters upon ligand binding. In particular, strands B3, B5, and B6, which have most of the residues involved in the dimer interaction, have the reduced order parameters in the steroid-bound KSI, indicating the increased high-frequency (pico- to nanosecond) motions in the intersubunit region of this homodimeric enzyme. Our results differ from those of previous studies on the backbone dynamics of monomeric proteins, in which high-frequency internal motions are typically restricted upon ligand binding.

Binding Sites↗
PubMed2000

Binding of 2-naphthols to D38E mutants of 3-oxo-Delta 5-steroid isomerase: variation of ligand ionization state with the nature of the electrophilic component.

3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of a variety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4) isomers. The mechanism involves sequential enolization and ketonization, with Asp-38 acting to transfer a proton from C-4 to C-6 through a dienol(ate) intermediate. We have previously proposed that this intermediate is anionic, with stabilization provided from direct hydrogen bonding from Tyr-14 and Asp-99 to the oxygen of the steroid. In this work, we analyze the binding of substituted 2-naphthols, which are analogues of the intermediate dienol, to the D38E KSI mutant and the corresponding double mutants lacking one of the two electrophilic groups (D38E/Y14F and D38E/D99A). The binding of these naphthols to the mutant KSIs at pH 7 is described by the modified Bronsted equation: log K(D) = alpha(pK(a)) + constant, where K(D) is the dissociation constant of the complex. The high value of alpha for D38E (alpha = 0.87 +/- 0.06) indicates that the negative charge in these D38E-naphthol complexes is localized almost exclusively on the bound ligand. In contrast, values of alpha for the double mutants (alpha = 0.28 +/- 0.02 for D38E/Y14F and alpha = 0.25 +/- 0.02 for D38E/D99A) are consistent with very little negative charge on the oxygen of the bound naphthol. Ultraviolet spectra of 5-nitro-2-naphthol and the fluorescence spectra of equilenin bound to these mutants support this interpretation. Extrapolation of these results to the intermediate in the catalytic reaction suggests that for the reaction with D38E, the intermediate is a negatively charged dienolate with hydrogen bonding from both Tyr-14 and Asp-99. Removal of either one of these H-bond donors (Tyr-14 or Asp-99) causes destabilization of the anion and results in a dienol enzyme-intermediate complex rather than a dienolate.

Aspartic Acid↗
PubMed2001

Ferrographic tracking of bacterial transport in the field at the narrow channel focus area, Oyster, VA.

The first results from an innovative bacterial tracking technique, ferrographic capture, applied to bacterial transport in groundwater are reported in this paper. Ferrographic capture was used to analyze samples during an October 1999 bacterial injection experiment at the Narrow Channel focus area of the South Oyster site, VA. Data obtained using this method showed that the timing of bacterial breakthrough was controlled by physical (hydraulic conductivity) heterogeneity in the vertical dimension as opposed to variation in sedimentsurface or aqueous chemical properties. Ferrographic tracking yielded results that compared well with results from other tracking techniques over a concentration range of 8 orders of magnitude and provided a low detection limit relative to most other bacterial tracking techniques. The low quantitation limit of this method (approximately 20 cells/mL) allowed observation of transport of an adhesion-deficient bacterium over distances greater than 20 m in the fine sand aquifer underlying this site.

Bacteria↗
PubMed2005

Spatial variation in deposition rate coefficients of an adhesion-deficient bacterial strain in quartz sand.

The transport of bacterial strain DA001 was examined in packed quartz sand under a variety of environmentally relevant ionic strength and flow conditions. Under all conditions, the retained bacterial concentrations decreased with distance from the column inlet at a rate that was faster than loglinear, indicating that the deposition rate coefficient decreased with increasing transport distance. The hyperexponential retained profile contrasted againstthe nonmonotonic retained profiles that had been previously observed for this same bacterial strain in glass bead porous media, demonstrating that the form of deviation from log-linear behavior is highly sensitive to system conditions. The deposition rate constants in quartz sand were orders of magnitude below those expected from filtration theory, even in the absence of electrostatic energy barriers. The degree of hyperexponential deviation of the retained profiles from loglinear behavior did not decrease with increasing ionic strength in quartz sand. These observations demonstrate thatthe observed low adhesion and deviation from log-linear behavior was not driven by electrostatic repulsion. Measurements of the interaction forces between DA001 cells and the silicon nitride tip of an atomic force microscope (AFM) showed that the bacterium possesses surface polymers with an average equilibrium length of 59.8 nm. AFM adhesion force measurements revealed low adhesion affinities between silicon nitride and DA001 polymers with approximately 95% of adhesion forces having magnitudes < 0.8 nN. Steric repulsion due to surface polymers was apparently responsible for the low adhesion to silicon nitride, indicating that steric interactions from extracellular polymers controlled DA001 adhesion deficiency and deviation from log-linear behavior on quartz sand.

Bacterial Adhesion↗
PubMed2006

Enhanced phenol biodegradation and aerobic granulation by two coaggregating bacterial strains.

The effect of coaggregation of the two bacterial strains Propioniferax-like PG-02 and Comamonassp. PG-08 on phenol degradation and aerobic granulation was investigated. While PG-02 was characterized as a phenol-degrader with a low half-saturation kinetics constant, PG-08 possessed strong aggregation ability with poor phenol degradation ability. The two strains coaggregated through involvement of lectin-saccharide interactions with the adhesin protein on strain PG-02 and the complementary sugar receptor on strain PG-08. Using the V. harveyi reporter strain BB170, it was found that both strains could produce autoinducer-2-like signals. If incubated together, the two strains showed cooperation for phenol degradation. In batch, the coculture degraded phenol at an initial concentration of 250 mg L(-1), faster than each strain separately. Bioaugmentation with simultaneously the two strains in sequencing batch reactors significantly improved phenol removal and aerobic granulation as compared to monoculture bioaugmentation. Bacterial coaggregation might be an integral component of the aerobic granulation process. Investigation of in situ occurrence of coggregation in aerobic granulation would help unveil its molecular mechanism. Then the granulation process could be improved through selection of specific microbial groups.

Biodegradation, Environmental↗
PubMed1999

Diversity and correlation of specific aromatic hydrocarbon biodegradation capabilities.

This work investigated the biodegradation capabilities of indigenous microorganisms exposed to different combinations of aromatic hydrocarbons. Considerable diversity was found in the catabolic specificity of 55 strains. Toluene was the most commonly degraded compound, followed by p-xylene, m-xylene and ethylbenzene. Strains capable of degrading o-xylene and benzene, which were the least-frequently-degraded compounds, exhibited broader biodegradation capabilities. Kappa statistics showed a significant correlation between the abilities to degrade toluene and ethylbenzene, p-xylene and m-xylene, and p-xylene and o-xylene. The ability to degrade naphthalene was correlated to the ability to degrade other alkylbenzenes, but not benzene. In addition, the inability to degrade benzene was correlated to the inability to degrade o-xylene. Factorial analysis of variance showed that biodegradation capabilities were generally broader when aromatic hydrocarbons were fed as mixtures than when fed separately. Beneficial substrate interactions included enhanced degradation of benzene, p-xylene, and naphthalene when toluene was present, and enhanced degradation of naphthalene by ethylbenzene. Such heuristic relationships may be useful to predict biodegradation patterns when bacteria are exposed to different aromatic hydrocarbon mixtures.

Bacteria↗
PubMed1999

Crystal structure of delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin settles the correct hydrogen bonding scheme for transition state stabilization.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp(38) (pK(a) approximately 4.7) was identified as the general base abstracting the steroid C4beta proton (pK(a) approximately 12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr(14) OH and Asp(99) COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp(99) donates the hydrogen bond to Tyr(14), which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr(14) OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.

Binding Sites↗
PubMed2001

Different mechanisms of the binding of soluble electron donors to the photosynthetic reaction center of Rubrivivax gelatinosus and Blastochloris viridis.

The tetraheme cytochrome subunits of the photosynthetic reaction centers (RCs) in two species of purple bacteria, Rubrivivax gelatinosus and Blastochloris (Rhodopseudomonas) viridis, were compared in terms of their capabilities to bind different electron-donor proteins. The wild-type RCs from both species and mutated forms of R. gelatinosus RCs (with amino acid substitutions introduced to the binding domain for electron-donor proteins) were tested for their reactivity with soluble cytochromes and high potential iron-sulfur protein. Cytochromes from both species were good electron donors to the B. viridis RC and the R. gelatinosus RC. The reactivity in the R. gelatinosus RC showed a clear dependence on the polarity of the charges introduced to the binding domain, indicating the importance of the electrostatic interactions. In contrast, high potential iron-sulfur protein, presumed to operate according to the hydrophobic mechanism of binding, reacted significantly only with the R. gelatinosus RC. Evolutionary substitution of amino acids in a region of the binding domain on the cytochrome subunit surface probably caused the change in the principal mode of protein-protein interactions in the electron-transfer chains.

Bacterial Proteins↗