Degradation of β-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

β-Hexachlorocyclohexane (β-HCH) is the most recalcitrant among the α-, β-, γ-, and δ-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of γ-HCH in Sphingomonas paucimobilis UT26, metabolizes β-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.     

Enantioselective Dehydrochlorination of δ-Hexachlorocyclohexane and δ-Pentachlorocyclohexene by LinA1 and LinA2 from Sphingobium indicum B90A

δ-Hexachlorocyclohexane (δ-HCH), one of the prevalent isomers of technical HCH, was enantioselectively dehydrochlorinated by the dehydrochlorinases LinA1 and LinA2 from Sphingobium indicum B90A to the very same δ-pentachlorocyclohexene enantiomer. Racemic δ-pentachlorocyclohexene, however, was transformed with opposite enantioselectivities by the two enzymes. A transformation pathway based on an anti-1,2-elimination, followed by a syn-1,4-elimination and a subsequent syn-1,2-elimination is postulated.     

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.     

Inhibition of α-, β-, γ-, and δ-carbonic anhydrases from bacteria and diatoms with N′-aryl-N-hydroxy-ureas

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with K_Is in the range of 97.5?nM – 7.26?µM and 52.5?nM – 1.81?µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (K_Is of 4.75 – 8.87?µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (K_Is?>?10?µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (K_Is of 59.8?nM – 6.42?µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (K_Is of 33.3?nM – 8.74?µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (K_Is?>?10?µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.     

Kinetic study of the quenching reaction of singlet oxygen by α-, β-, γ-, δ-tocotrienols,and palm oil and soybean extracts in solution

Measurements of the singlet oxygen (¹O₂) quenching rates (k_Q (S)) and the relative singlet oxygen absorption capacity (SOAC) values were performed for 11 antioxidants (AOs) (eight vitamin E homologues (α-, β-, γ-, and δ-tocopherols and -tocotrienols (-Tocs and -Toc-3s)), two vitamin E metabolites (α- and γ-carboxyethyl-6-hydroxychroman), and trolox) in ethanol/chloroform/D₂O (50:50:1, v/v/v) and ethanol solutions at 35?°C. Similar measurements were performed for five palm oil extracts 1–5 and one soybean extract 6, which included different concentrations of Tocs, Toc-3s, and carotenoids. Furthermore, the concentrations (wt%) of Tocs, Toc-3s, and carotenoids included in extracts 1–6 were determined. From the results, it has been clarified that the ¹O₂-quenching rates (k_Q (S)) (that is, the relative SOAC value) obtained for extracts 1–6 may be explained as the sum of the product {Σ k_Q^AO-i (S) [AO-i]/100} of the rate constant (k_Q^AO-i (S)) and the concentration ([AO-i]/100) of AO-i (Tocs, Toc-3s, and carotenoid) included.     

The ABRF-MIRG’02 Study: Assembly State,Thermodynamic, and Kinetic Analysis of an Enzyme/Inhibitor Interaction

Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.     

Characterization of Glyoxalase I (E. coli)—Inhibitor Interactions by Electrospray Time-of-Flight Mass Spectrometry and Enzyme Kinetic Analysis

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.     

Allelic Variants Interaction of CLOCK Gene and G‐Protein β3 Subunit Gene with Diurnal Preference

The 3111 C/T single nucleotide polymorphism (SNP) in the CLOCK gene and the 825C/T SNP in the G‐protein β3 subunit gene (GNB3) have been reported to influence diurnal preference. This study has attempted to characterize the association between the CLOCK gene and GNB3 polymorphisms and diurnal preference in healthy Korean college students. All subjects completed the 13‐item Composite Scale for Morningness (CSM). The interaction between the 3111 C/T SNP in the CLOCK gene and the 825 C/T SNP in the GNB3 gene significantly influenced diurnal preference, according to the CSM Performance subscore (F=10.94, p=0.001). However, when the different polymorphisms of the two genes were analyzed independently, no direct correlations with diurnal preference were detected. The CLOCK gene 3111 C/T SNP and GNB3 gene 825 C/T SNP were found to manifest a gene‐gene interaction that affects diurnal preference.     

Kinetic behaviour and specificity of β-fructosidases in the hydrolysis of plant and microbial fructans

Fructosidases, in particular exo-β-fructosidases, may act on fructans such as inulins and levans of plant and bacterial origin to produce fructose. In this paper, the kinetic properties of a commercial preparation (Fructozyme L) and a recombinant exoinulinase (BfrA) from Thermotoga maritima, were studied using fructan polymer substrates from various sources. Both enzymatic preparations preferentially hydrolyzed β2-1 linkages and low molecular weight fructans. We show that chicory inulin is degraded most efficiently by both preparations, followed by bacterial inulin, in spite of its high molecular weight and branching in β2-6 positions. All bacterial levans were more slowly hydrolyzed. Michaelis–Menten kinetics describe the hydrolysis of sucrose and low molecular weight fructans (≤8.3 kDa) by both enzyme preparations, while first order kinetics were observed with respect to bacterial fructans due to the high molecular weight and, therefore, low molar concentrations. Comparison of second order rate constants indicates that bacterial inulin (Leuconostoc citreum CW28) is hydrolyzed more slowly with both enzyme preparations than chicory inulin by approximately one order of magnitude. For Leuconostoc mesenteroides NRRL B-512F levan, the second order rate constant for Fructozyme L is 200-fold lower than for chicory inulin. However, the second order rate constant for BfrA is only 22-fold lower than for chicory inulin. Taken together, our studies characterize the kinetics of fructan hydrolysis and also suggest that the kinetic parameters may be used to differentiate between fructan structures.     

Kinetic mechanism of active site assembly and chemical catalysis of DNA polymerase β

It has been inferred from structural and computational studies that the mechanism of DNA polymerases involves subtle but important discrete steps that occur between binding and recognition of the correct dNTP and chemical catalysis. These steps potentially include local conformational changes involving active site residues, reorganization of Mg(2+)-coordinating ligands, and proton transfer. Here we address this broad issue by conducting extensive transient state kinetic analyses of DNA polymerase β (Pol β). We also performed kinetic simulations to evaluate alternative kinetic models. These studies provide some support for two-step subdomain closing and define constraints under which a kinetically significant prechemistry step can occur. To experimentally identify additional microscopic steps, we developed a stopped flow absorbance assay to measure proton formation that occurs during catalysis. These studies provide direct evidence that formation of the enzyme-bound 3'-O(-) nucleophile is rate determining for chemistry. We additionally show that at low pH the chemical step is rate limiting for catalysis, but at high pH, a postchemistry conformational step is rate limiting due to a pH-dependent increase in the rate of nucleotidyl transfer. Finally, we performed exhaustive analyses of [Mg(2+)] and pH effects. In contrast to published studies, the results suggest an irregular pH dependence of k(pol), which is consistent with general base catalysis involving cooperativity between two or more protonic residues. Overall, the results represent significant advancement in the kinetic mechanism of Pol β and also reconcile some computational and experimental findings.     

Attempt at Affinity Labeling of α- and β- Amylases by α- and β-d-Glucopyranosides and α- and β-Maltooligosaccharides with 2,3-Epoxypropyl Residue as Aglycone: Specific Inactivation of β-Amylases

Among 2,3-epoxypropyl α-d-glucopyranoside and 2,3-epoxypropyl α-maltooligosaccharides and the β-anomers, 2,3-epoxypropyl α-d-glucopyranoside (α-EPG) strongly inactivated the β-amylases [EC 3.2.1.2] of sweet potato, barley, and Bacillus, cereus, in addition to soybean β amylase [J. Biochem., 99, 1631 (1986)]. However, none of the compounds used inactivated any α-amylases [EC 3.2.1.1] of porcine pancreas, Aspergillus oryzae, or Bacillus amyloliquefaciens. Irreversible incorporation of ¹⁴C-labeled α-EPG into β-amylases was stoichiometric, i.e., one α-EPG per active site of the enzyme was bound, and the inactivations were almost complete. The results suggest that α-EPG is an affinity labeling reagent selective for β-amylase. Slow inactivations by the other compounds were also observed, depending on the difference of source of β amylase.     

Characterization of Four Novel Variants of Goat βA-Globin Gene

Four novel alleles of the adult -globin gene of Capra hircus were observed in an extended study on hemoglobin polymorphism in goat breeds living in the island of Sardinia. Nucleotide sequencing showed that one of these alleles is due to a 2 bp substitution at codon 125 ( G, "LeuGlu). Two substitutions, the silent CT for Leu at codon 78 and the conservative A G (Lys Arg) at codon 104, are shared by the other three alleles, two of them having additional mutations, which suggests a common origin. The allele we provisionally called the ^Y shares four out of five amino acid substitutions, together with the same polymorphisms in the IVSII, we observed previously in the rather common ^E gene. This evidence allowed the origin of the ^E gene to be better characterized. The data increase to seven the number of alleles at the goat ^A -globin locus characterized thus far at the molecular level. A simplified nomenclature for the increasing number of goat -globin alleles is presented.     

Analysis of the paired TCR α- and β-chains of single human T cells

Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV) and multiple sclerosis (MS). In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8(+) T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8(+) T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity.     

Kinetic Fluorescence Spectral Analysis of Core Antennas CP43 and CP47 of Photosystem Ⅱ with Ultrafast Time-resolved Technology

Ultrafast time-resolved fluorescence experiments have been performed with core antennas CP43 and CP47 of PS Ⅱ. Their dynamic fluorescence spectra were obtained with excitation wavelength 514.5 nm. For CP43, the emission spectrum was found to be from 640 to 780 nm with a peak at ～680 nm and the lifetime of fluorescence was 3.54 ns. For CP47, the emission spectrum was from 630 to 775 nm with a peak at ～691 nm and the fluorescence lifetime was 3.22 ns. The fluorescence emission efficiencies of Chl a in CP43 and CP47 were calculated to be approximately 38.3% and 40.6%, respectively. The energy transfer from β-Car to Chl a in CP43 and CP47 was discussed. The rates of energy transfer from β-Car to Chl a were measured to be about 9.6×1011 s-1 and 1.3×1012 s-1 and energy transfer efficiencies 47.5% and 66.5% respectively. The edge-edge distances between β-Car and Chl a in CP43 and CP47 were estimated to be ～0.110 nm and ～0.085nm respectively.     

Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms and Escherichia coli

Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.Indicators of pollution (e.g., coliforms, fecal coliforms, and Escherichia coli) are traditionally used for monitoring the microbiological safety of water supplies and recreational water. Several techniques for detection of coliforms and E. coli are based on enzymatic hydrolysis of fluorogenic or chromogenic substrates for β-d-galactosidase and β-d-glucuronidase (9, 20). Current methods of recovery are usually culture based, and the analysis time is 18 to 24 h. In addition to enzymatic activity, these techniques use growth at appropriate temperatures in the presence of inhibitors, combined with demonstration of enzymatic activity, to selectively detect target bacteria.Rapid methods which require less than 6 h and are based on chromogenic, fluorogenic, or chemiluminogenic substrates for detection of coliforms, fecal coliforms, or E. coli have been described (1–3, 10, 27, 28). These rapid assays are based on the assumption that β-d-galactosidase and β-d-glucuronidase are markers for coliforms and E. coli, respectively. However, when the incubation time is 1 h or less, growth is not a selective step, and all β-d-galactosidase-positive or β-d-glucuronidase-positive microorganisms in a water sample contribute to the activity measured. At low initial concentrations of target bacteria (i.e., E. coli and total coliforms), increasing the preincubation time to 5 to 6 h did not result in a predominance of target bacteria compared to nontarget bacteria (28).The β-d-galactosidase or β-d-glucuronidase activity calculated per cultivable coliform or fecal coliform bacterium in environmental samples can be 1 to 2 log units higher than the activity per induced E. coli cell in pure culture (11, 26). The presence of active, noncultivable bacteria can be one reason for this. Studies of survival (7, 24, 25) and disinfection (26) of E. coli have shown that loss of cultivability does not necessarily result in a loss of β-d-galactosidase activity. The presence of false-positive bacteria can be another reason.β-d-Galactosidase has been found in numerous microorganisms, including gram-negative bacteria (e.g., strains belonging to the Enterobacteriaceae, Vibrionaceae, Pseudomonadaceae, and Neisseriaceae), several gram-positive bacteria, yeasts, protozoa, and fungi (17, 29). β-d-Glucuronidase is produced by most E. coli strains and also by other members of the Enterobacteriaceae, including some Shigella and Salmonella strains and a few Yersinia, Citrobacter, Edwardia, and Hafnia strains. Production of β-d-glucuronidase by Flavobacterium spp., Bacteroides spp., Staphylococcus spp., Streptococcus spp., anaerobic corynebacteria, and Clostridium has also been reported (12).High numbers of false-positive bacteria in sewage and contaminated water have been revealed by enumeration of β-d-galactosidase- and β-d-glucuronidase-positive CFU on nonselective agar supplemented with fluorogenic or chromogenic substrates (11, 28). Whether the activity from nontarget organisms can be neglected in a rapid assay depends on the number of nontarget organisms compared with the number of target bacteria and also on the level of their enzyme activity. Plant and algal biomass must be present at high concentrations to interfere in rapid bacterial β-d-galactosidase and β-d-glucuronidase assays (8).The main objective of this study was to investigate the enzyme characteristics of β-d-galactosidase- and β-d-glucuronidase-positive bacteria isolated from environmental water samples and to evaluate the potential influence of false-positive bacteria in rapid assays for coliform bacteria or E. coli in water. The effect of temperature on enzyme activity and on the interference of nontarget bacteria in the rapid assays was investigated as an important factor.(Some of the results were presented at the 97th General Meeting of the American Society for Microbiology 1997, Miami Beach, Fla., 4 to 8 May 1997.)     

Genetic Analysis of the First 4 Patients with β-Ureidopropionase Deficiency

β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyses the irreversible hydrolysis of N-carbamyl-ß-aminoisobutyric acid or N-carbamyl-ß-alanine to β-aminoisobutyric acid or ß-alanine, ammonia, and CO₂. Analysis of the β-ureidopropionase gene (UPB1) of the first 4 patients presenting with a complete enzyme deficiency, revealed the presence of 2 splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). RT-PCR analysis of the complete β-ureidopropionase cDNA suggested that both splice-site mutations lead to a variety of alternative splice variants, with deletions of a single or several exons. The alanine at position 85 was not conserved in other eukaryotic β-ureidopropionase protein sequences.