Hydraulic conductivity assessment of falling head percolation tests used for the design of on-site wastewater treatment systems.

The suitability of a location for an on-site wastewater treatment process (for areas which lack access to centralised wastewater treatment systems) requires an assessment of the permeability of the soil into which the effluent will be discharged. In many jurisdictions this is determined using some type of in-situ percolation test. Falling head percolation tests, which give a value of percolation time (PT) that is empirically related to the notion of hydraulic conductivity, are widely used as they are relatively simple to carry out, but the test does not have a sound theoretical framework and test methods are not standardised internationally. In comparison, the saturated hydraulic conductivity of a soil obtained from a constant head well permeameter test is independent of test conditions, and so is a more suitable metric for design. A database of over 900 falling head tests carried out across a range of different subsoil types in Ireland has been collated, all with the inherent limitations of the existing regulative framework regarding the percolation test and soil texture assessment. These tests were then modelled using Hydrus 2-D numerical modelling simulations to determine equivalent field saturated hydraulic conductivity (Kfs) values and thereby provide a correlation with PT values across the range of subsoil conditions. In addition, falling head tests have been carried out in parallel to constant head permeameter tests in the field and compared against the relationship derived from the broad dataset of simulated results. This revealed an optimal solution by which to determine Kfs from the field permeameter test (using parameters recommended for most structured soils from clays to loams). The trendline based on Irish data was also compared against more generic formulations of the relationship between PT, and Kfs and shown to match closely, particularly the Reynolds (2016) 'unified' methodology. Finally, the Irish threshold PT limits for on-site wastewater treatment have been converted to Kfs values and compared against other international standards.

YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase.

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N-Calpha of Gly734. A recombinant protein comprising the core of PFL (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with PFL activase a heterooligomeric PFL enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for PFL's glycyl radical domain, utilized for rapid recovery of PFL activity (and thus ATP generation) in cells that have experienced oxidative stress.

Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism.

The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969-975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)). Using [1-(14)C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.

Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase.

Pyruvate formate-lyase (PFL) from Escherichia coli uses a radical mechanism to reversibly cleave the C1-C2 bond of pyruvate using the Gly 734 radical and two cysteine residues (Cys 418, Cys 419). We have determined by X-ray crystallography the structures of PFL (non-radical form), its complex with the substrate analog oxamate, and the C418A,C419A double mutant. The atomic model (a dimer of 759-residue monomers) comprises a 10-stranded beta/alpha barrel assembled in an antiparallel manner from two parallel five-stranded beta-sheets; this architecture resembles that of ribonucleotide reductases. Gly 734 and Cys 419, positioned at the tips of opposing hairpin loops, meet in the apolar barrel center (Calpha-Sgamma = 3.7 A). Oxamate fits into a compact pocket where C2 is juxtaposed with Cys 418Sgamma (3.3 A), which in turn is close to Cys 419Sgamma (3.7 A). Our model of the active site is suggestive of a snapshot of the catalytic cycle, when the pyruvate-carbonyl awaits attack by the Cys 418 thiyl radical. We propose a homolytic radical mechanism for PFL that involves Cys 418 and Cys 419 both as thiyl radicals, with distinct chemical functions.

A dehydroalanyl residue can capture the 5'-deoxyadenosyl radical generated from S-adenosylmethionine by pyruvate formate-lyase-activating enzyme.

The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is produced post-translationally by pyruvate formate-lyase-activating enzyme (PFL activase), employing adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates. Previous 2H-labelings found incorporation of the pro-S hydrogen of Gly-734 into the 5'-deoxyadenosine co-product, indicating that a deoxyadenosyl radical intermediate, generated by reductive cleavage of AdoMet, serves as the actual H atom abstracting species in this system. We have now examined an octapeptide (Suc-Arg-Val-Pro-DeltaAla-Tyr-Ala-Val-Arg-NH2) that is analogous to the Gly-734 site of the PFL polypeptide but contains a dehydroalanyl residue (DeltaAla) in the glycyl position. Applied to the PFL activase reaction, this peptide becomes C-adenosylated at the olefinic beta carbon of DeltaAla. The modified peptide was isolated in micromol-quantities and characterized, after chymotryptic truncation, by MS and 2D NMR. PFL activase functions catalytically (kcat >/= 1 min-1) in the peptide modification reaction, which occurs with stoichiometric consumption of AdoMet. The mechanism appears to involve addition of the nucleophilic deoxyadenosyl radical to the electrophilic CC double bond of DeltaAla, followed by quenching of the peptide backbone-centered adduct radical by the buffer medium. The trapping-property of the DeltaAla residue should be exploitable in investigating of how the Fe4S4 protein PFL activase generates the highly reactive deoxyadenosyl radical.

Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form.

The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5'-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform. The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form. The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S]+) from axial to rhombic symmetry.

Adenosylmethionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen atom. Studies of [2H]glycine-substituted enzyme and peptides homologous to the glycine 734 site.

The active form of pyruvate formate-lyase (PFL) from Escherichia coli contains a glycyl radical in position 734 of the polypeptide chain which is produced post-translationally by pyruvate formate-lyase-activating enzyme (PFL activase) using S-adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates (Wagner, A.F. V., Frey, M., Neugebauer, F.A., Schäfer, W., and Knappe, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 996-1000). Studying radical synthesis with [2-2H]glycine-labeled PFL, we have now found stoichiometric incorporation of a 2H atom into the 5'-deoxyadenosine (dAdo) co-product via mass and NMR spectroscopic analyses. Furthermore, a series of peptides homologous to the Gly-734 site of PFL have been synthesized for analyzing recognition determinants of PFL activase. Peptides that proved active as substrates (monitored by [14C]dAdo formation from [14C]AdoMet) were also competitive inhibitors of PFL conversion to the radical form. In the sequence of the standard peptide Arg-Val-Ser-Gly-Tyr-Ala-Val, which corresponds to amino acid residues 731-737 of PFL, the Gly residue was replaceable by D-Ala (actually displaying enhanced efficiency), whereas a normal Ala totally abolished the interaction with PFL activase. Our results show that the radical in pyruvate formatelyase is produced by stereospecific abstraction of the pro-S hydrogen of glycine 734 by the 5'-dAdo radical generated in the active center of PFL activase. Gly-734 is probably located in a beta-turn segment of the protein.

Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli.

The AdhE protein of Escherichia coli is a homopolymer of 96-kDa subunits harboring three Fe(2+)-dependent catalytic functions: acetaldehyde-CoA dehydrogenase, alcohol dehydrogenase, and pyruvate formatelyase (PFL) deactivase. By negative staining electron microscopy, we determined a helical assembly of 20-60 subunits into rods of 45-120 nm in length. The subunit packing is widened along the helix axis when Fe2+ and NAD are present. Chymotrypsin dissects the AdhE polypeptide between Phe762 and Ser763, thereby retaining the alcohol dehydrogenase activity on the NH2-terminal core, but destroying all other activities. PFL deactivation, i.e. quenching of the glycyl radical in PFL by the AdhE protein, was examined with respect to cofactor involvements (Fe2+, NAD, and CoA). This process is coupled to NAD reduction and requires the intact CoA sulfhydryl group. Pyruvate and NADH are inhibitors that affect the steady-state level of the radical form of PFL in a reconstituted interconversion cycle. Studies of cell cultures found that PFL deactivation in situ is initiated at redox potentials of greater than or equal to +100 mV. Our results provide insights into the structure/function organization of the AdhE multienzyme and give a rationale for how its PFL radical quenching activity may be suppressed in situ to enable effective glucose fermentation.

The free radical in pyruvate formate-lyase is located on glycine-734.

Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models. N-coupling was verified through uniform 15N-labeling. The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.

Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE.

A 4.8 kb DNA-fragment was cloned and sequenced encompassing the structural gene of PFL-deactivase (2.7 kb) and 2 kb of the 5' flanking region that contains the elements for anaerobic induction. A mutant lacking deactivase was shown to require exogenous electron acceptors for anaerobic growth with glucose. This revealed the identity of PFL-deactivase with the alcohol and acetaldehyde dehydrogenases of E. coli. The multienzyme represents a homopolymeric protein (approximately 40 x 96 kDa) requiring Fe2+ for all functions.

A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli.

Anaerobically growing Escherichia coli cells contain the enzyme pyruvate formate-lyase which catalyses the non-oxidative cleavage of pyruvate to acetyl-CoA and formate. The enzyme is subject to interconversion between inactive and active forms. The active form contains an oxygen-sensitive organic free radical located on the polypeptide chain which is essential for catalysis. It affords a novel homolytic C-C bond cleavage of the pyruvate substrate. The radical is generated by an iron-dependent converter enzyme which requires reduced flavodoxin and adenosyl methionine as co-substrates and pyruvate as a positive allosteric effector. A second converter enzyme, also iron-dependent, accomplishes the removal of the radical. This post-translational interconversion cycle controls the activity state of pyruvate formate-lyase in the anaerobic cell. Anaerobic conditions also regulate pyruvate formate-lyase at the level of gene expression. Multiple promoters are responsible for effecting a twelve to fifteen fold induction and they are coordinately controlled in response to the oxygen and metabolic status of the cell by sequences which are located far upstream of the pfl coding region. The transcription factor Fnr has been identified as being responsible for part of the anaerobic control of pfl expression, probably through direct interaction with the upstream sequences. In contrast, the expression of the gene encoding the first iron-dependent converter enzyme is unaffected by anaerobiosis and is independent of the Fnr protein.

[The scientific basis and evaluation of the present-day concept of risk factors and their significance for the prevention of chronic non-communicable diseases]. / Zur wissenschaftlichen Basis bzw. zur Absicherung des gegenwärtigen Risikofaktorenkonzepts und seiner Bedeutung für die Prävention chronischer nichtübertragbarer Erkrankungen.

Chronic diseases increased in their significance, to them first of all belong cardiovascular diseases. They particularly restrict a further increase of the life-expectancy of the persons older than 40 years. In the judgement of the development of these groups of diseases during the last decades the so-called concept of risk factors was developed, which obtained particular significance for the myocardial infarction and the chronic cardiovascular diseases. Though the epidemiologic investigations up to now could cover only correlations and not causal connections, the latter - that means causal conditions - seem to get an increasing support by results of the adequate basic research. Still more impressionable is, however, the regression of the mortality due to cardiovascular diseases which took place during recent years in connection with the changes of the living habits in several countries of the earth. From the results of our own research the particular urgencies concerning the preventive medicine in our country are described which must consist in changes of the living habits, in particular of the habits of nutrition and the abuse of coffee, tea, tobacco, alcohol, etc., moreover also in personal behaviour. Independent of this genetic factors certainly play a decisive part. Apart from the individual strategy mass-strategic aspects will obtain a greater importance in the following years.

The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite.

The first-derivative EPR spectrum of the active form of Escherichia coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site. The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formate-lyase which destroys the enzyme radical. Protein-linked 1-hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445-450]. EPR spectroscopy of this system has now identified the corresponding alpha-phosphoryl radical as a reaction intermediate [g = 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0 degrees C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.

[Dose-reduced antihypertensive agents--use in complex nonmedicamentous therapy of hypertension]. / Dosisreduzierte Antihypertensiva--Anwendung bei komplexer nichtmedikamentöser Hochdrucktherapie.

In order to estimate the influence of a non-medicamentous therapy (CNT) on the consumption of medicaments and coronary risk in high blood pressure 73 hypertensives of a medicamentously stabilized CNT-group were examined in comparison to a group of the same size of patients with hypertension who were managed exclusively medicamentously for behaviour of blood pressure, need of antihypertensive drugs and changes of hypertension-associated risk factors. After an exactly controlled 6-month treatment hypertensives with additionally recommended far-reaching CNT showed an economization of medicaments by scarcely the half in comparison to the reference group. By means of suitable control methods a causal non-medicamentously conditioned decrease of blood pressure could be excluded. A different need of antihypertensive drugs was simulated by the exacter intake of medicaments in the index-patients. Notwithstanding the metabolic effects of the additional therapy have induced a positive change of atherogenic lipids. The examinations indicate in general the difficulty of the judgement of efficacy of non-medicamentous therapeutic measures in connection with a rational dose-reduced long-term therapy with antihypertensive drugs.