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H Y Neujahr

Publications and source records attributed to H Y Neujahr.

At least 37 records · Page 2Linked to original sources
PubMed1980

Phenol hydroxylase from yeast: a lysyl residue essential for binding of reduced nicotinamide adenine dinucleotide phosphate.

The inducible enzyme phenol hydroxylase from Trichosporon cutaneum is a FAD-containing monooxygenase which catalyzes the NADPH-dependent hydroxylation of phenol to catechol. The enzyme contains 16 cysteinyl residues, 6--8 of which are essential for retention of FAD and for activity. The complete amino acid composition is now reported as well as the results of studies with amino group reagents. A number of amino group reagents inhibit the enzyme severely, most of them with a concomitant, more or less extensive release of FAD. P-pyridoxal inhibits the enzyme specifically, without affecting its FAD content. The P-pyridoxal modified enzyme has a characteristic absorption peak at 325 nm indicating the presence of a N epsilon-pyridoxyllysyl derivative. Such a derivative was identified in hydrolysates of the modified enzyme by means of column chromatography. The results obtained with P-pyridoxal-modified enzyme indicate that a lysyl residue is essential for activity by being involved in binding of the co-substrate NADPH. These results are corroborated by kinetic studies showing competition between P-pyridoxal and NADPH for the binding site. The reactivity of the essential lysyl residue toward P-pyridoxal is significantly increased in the presence of phenol. Inhibition by excess phenol shifts toward lower concentrations in the presence of P-pyridoxal. On the basis of the present results together with previous findings, we propose that phenol acts as a substrate effector by causing a conformation change which exposes a reactive lysyl residue with a concomitant burying of the essential SH groups and a tighter attachment of FAD.

Amino Acids↗
PubMed1980

cis,cis-Muconate cyclase from Trichosporon cutaneum.

The inducible enzyme catalysing the conversion of cis,cis-muconate to (+)-muconolactone was purified 300-fold from the yeast Trichosporon cutaneum, grown on phenol. The enzyme has a sharp pH optimum at pH 6.6. It reacts also with several monohalogen derivatives and with one monomethyl derivative of cis,cis-muconate, but not with cis,trans- or trans,trans-muconate or 3-carboxy-cis,cis-muconate. In contrast with the corresponding enzymes in bacteria, the yeast enzyme does not require added divalent metal ions for activity and is not inhibited by EDTA. The purified enzyme can be resolved into two peaks by isoelectric focusing. The two forms have pI 4.58 (cis,cis-muconate cyclase I) and pI 4.74 (cis, cis-muconate cyclase II), respectively. Each of these is homogenous on polyacrylamide-gel electrophoresis in the absence or presence of sodium dodecyl sulphate. The two enzyme forms have the same molecular weight (50000) as determined by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. They have the same Km value (25 microM) for cis,cis-muconate. They differ with respect to their content of free thiol groups. cis, cis-Muconate cyclase I contains one thiol group, essential for activity, but relatively stable upon storage. cis, cis-Muconate cyclase II contains two thiol groups that are readily oxidized during storage with concomitant loss of activity.

Cations, Divalent↗
PubMed1980

Maleylacetate reductase from Trichosporon cutaneum.

The enzyme catalysing the reduction of maleylacetate to 3-oxoadipate was purified 150-fold from Trichosporon cutaneum, induced for aromatic metabolisms by growth with resorcinol as a major carbon source. The enzyme separated upon electrofocusing into three species with PI values 4.6, 5.1 and 5.6. They had similar catalytic properties and the same molecular weight.

Chemical Phenomena↗
PubMed1980

Enzyme electrode for phenol.

An enzyme electrode is described for quantitative determination of phenol at micromolar concentrations. Immobilized phenol hydroxylase is attached to the surface of a Clark oxygen electrode. The maximum rate of oxygen consumption is linearly dependent on phenol concentration over the 0.5-50 microM range. The electrode can be used for at least 150 assays without an activity loss. Readout is very rapid--within 30 sec of sample addition. The electrode response is independent of pH between pH 6.5 and 9.5. The response increases linearly with temperature in the interval 10-40 degrees C. It is necessary to incubate the enzyme electrode in a buffer containing NADPH for a few minutes before the addition of sample. This is to make the electrode response independent of the diffusion rate of this cosubstrate. This and other diffusional effects on the performance of the phenol electrode are discussed.

Buffers↗
PubMed1979

Metabolism of phenol and resorcinol in Trichosporon cutaneum.

Trichosporon cutaneum was grown with phenol or resorcinol as the carbon source. The formation of beta-ketoadipate from phenol, catechol, and resorcinol was shown by a manometric method using antipyrine and also by its isolation and crystallization. Metabolism of phenol begins with o-hydroxylation. This is followed by ortho-ring fission, lactonization to muconolactone, and delactonization to beta-ketoadipate. No meta-ring fission could be demonstrated. Metabolism of resorcinol begins with o-hydroxylation to 1,2,4-benzenetriol, which undergoes ortho-ring fission yielding maleylacetate. Isolating this product leads to its decarboxylation and isomerization to trans-acetylacrylic acid. Maleylacetate is reduced by crude extracts to beta-ketoadipate with either reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate as a cosubstrate. The enzyme catalyzing this reaction was separated from catechol 1,2-oxygenase, phenol hydroxylase, and muconate lactonizing enzyme on a diethyl-aminoethyl-Sephadex A50 column. As a result it was purified some 50-fold, as was the muconate-lactonizing enzyme. Methyl-, fluoro-, and chlorophenols are converted to a varying extent by crude extracts and by purified enzymes. None of these derivatives is converted to maleylacetate, beta-ketoadipate, or their derivatives. Cells grown on resorcinol contain enzymes that participate in the degradation of phenol and vice versa.

Adipates↗
PubMed1975

Phenol hydroxylase from yeast. Sulfhydryl groups in phenol hydroxylase from Trichosporon cutaneum.

Thiol groups in phenol hydroxylase were measured using two different -SH reagents and amino acid analysis. Stepwise blocking of the -SH groups was correlated with enzyme activity and FAD content. The results indicate that the enzyme contains 16 -SH groups per molecule of Mr 1.48 X 10(5). At least four -SH groups are not accessible without the use of a denaturing agent. There is seemingly no disulphide bridge. On the whole, the reactivity towards p-hydroxymercuribenzoate is much greater than towards 5,5'-dithio-bis(2-nitrobenzoic acid). The two reagents seem to have a different specificity with respect to which -SH groups they attack. Either reagent dislocates FAD from the holoenzyme, leaving a characteristic mercaptide derivative of the apoenzyme. Such derivatives were used to prepare the apoenzyme. The -SH groups in the apoenzyme are much more reactive towards 5,5'-dithio-bis(2-nitrobenzoic acid) than the -SH groups in the holoenzyme. The stoichiometry of the reaction with 5,5'-dithio-bis(2-nitrobenzoic acid) indicates that at least 8 -SH groups are located in spatially close pairs. The most reactive pair of all does not appear to be of importance for enzyme activity. The two subsequent -SH pairs are essential for enzyme activity are are involved in FAD attachment. The reactivity of the -SH groups decreases dramatically in the presence of substrate, even at substrate concentrations equivalent to the level of the catalytic sites. The isolated apoenzyme has a tendency to aggregate. A large proportion of -SH groups in such aggregate(s) is buried, especially when EDTA is not used throughout the preparation of the apoenzyme. The aggregates are enzymically inactive.

Apoenzymes↗
PubMed1975

Lysis of modified walls from Lactobacillus fermentum.

The N and O substitution in wall peptidoglycan from Lactobacillus fermentum was studied in relation to growth phase, as well as the lytic activities and the effect of trypsin on them. The N-nonsubstituted sites were determined by dinitrophenylation techniques. The results indicate that an extensive substitution at the O groups takes place as cells go into the stationary growth phase, concomitant with a decrease in their lysozyme sensitivity. N-nonsubstituted residues, mainly glucosamine, occurred in both exponential-phase and stationary-phase walls but not in the corresponding peptidoglycans. Small amounts of N-nonsubstituted muramic acid were detected in walls and peptidoglycan from cells in the stationary growth phase only. N acetylation of isolated walls did not increase their lysozyme sensitivity but rather decreased it. Autolysis of walls was completely inhibited by the chemical modifications used. Trypsin stimulates the lysozyme sensitivity of native walls but has no effect on walls that had been O deacetylated and N acetylated. It is suggested that the effect of trypsin is due to its action as an esterase removing the O acetylation in lysozyme-resistant walls.

Acetylation↗
PubMed1975

Ultrastructure of Lactobacillus fermentum during early and late growth phases and during thiamine deficiency.

Thin sections of exponentially growing and stationary-phase cells of L. fermentum from thiamine sufficient and thiamine deficient media were studied by electron microscopy. Compared to the exponential-phase cells the stationary-phase cells from both types of media had thicker cell walls and cross walls and fewer and smaller granules of storage material. Exponential-phase, thiamine deficient cells had rather thin cell walls and small mesosomes.

Cell Membrane↗
PubMed1973

Factors affecting the resistance of Lactobacillus fermenti to lysozyme.

The sensitivity of Lactobacillus fermenti ATCC 9338 to lysozyme has its peak during the exponential phase of growth, after the autolytic activity of the organism has begun to decline. Cells from the stationary growth phase are resistant to lysozyme. The two lytic activities require different ionic conditions for their functioning; they appear mutually exclusive. Incubation with trypsin renders cells from all growth phases sensitive to lysozyme. The effect of trypsin is independent of the presence of lysozyme and vice versa, as demonstrated by use of trypsin inhibitors. Cells from early stationary phase of growth require higher temperature for optimum lysozyme action than do those from the exponential growth phase.

Aging↗
PubMed1971

CELL WALL AND PEPTIDOGLYCAN FROM Lactobacillus fermenti.

Cell walls from Lactobacillus fermenti were prepared by differential centrifugation of disrupted cells, with and without trypsin treatment. Approximately 16% of the dry weight of walls was found in a crude trichloroacetic acid extract of the walls; half of this amount remained upon further purification. The purufied extract lacked alanine, but contained substantial amounts of glucosamine. The walls constituted 23 to 33% of the dry weight of the cell. The chemical composition of the various types of wall preparations and of the peptidoglycan from them was studied. The peptidoglycan contained equimolar proportions of glucosamine, muramic acid, l-alanine, d-glutamic acid, and lysine, with somewhat lower proportions of d-aspartic acid and d-alanine. The chemical composition of the peptidoglycan is similar to that reported for three other lactobacilli. In addition to the major constituents of walls and peptidoglycan, there were several minor amino acids. The protein and the amounts of the minor amino acids decreased, and among these threonine and arginine were completely absent from preparations obtained with trypsin. Such preparations contained higher proportions of the d-isomers of alanine, glutamic acid, and aspartic acid as compared to walls and peptidoglycan prepared without trypsin. In addition, walls isolated with the use of trypsin were susceptible to lysozyme, whereas those prepared without trypsin were not. However, the trypsin treatment did not result in any change of the ultrastructure as revealed by electron microscope studies.

Amino Acids↗