Zur chemischen Zusammensetzung der Zellwand der Streptokokken

Zusammenfassung Das Murein (Peptidoglycan) eines aus Faeces isolierten Streptococcus, der in den wichtigsten Merkmalen mit Peptostreptococcus evolutus (Prevot) Smith übereinstimmt, weist folgende Molverhältnisse auf (aufgerundete bzw. abgerundete Zahlen): Mur:GlcNH₂:Ala:Glu:Lys:Gly=1:1:3:1:1:1. Das Verhältnis l-Alanin:d-Alanin=2,15:1. Die Glutaminsäure liegt in der d-Konfiguration und als Amid vor.Durch die Partialhydrolyse der Zellwände und die anschließende Isolierung und Identifizierung der Peptide konnte die Aminosäuresequenz des Mureins geklärt werden. Das Tetrapeptid stimmt mit der üblichen Sequenz l-Ala-d-Glu-NH₂-l-Lys-d-Ala der meisten übrigen Bakterien überein. Die Quervernetzung des Mureins wird durch das Peptid Glycyl-l-Alanin hergestellt, wobei l-Alanin an die -Aminogruppe des Lysins gebunden ist. Die Dinitrophenylierung der Zellwand ergab, daß 35% des Glycins und 6% des Lysins eine freie Aminogruppe aufweisen. Die Quervernetzung ist demnach nur zu höchstens 60% durchgeführt.

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The chemical composition of the cell walls of Streptococci III. The amino acid sequence of a glycine containing murein from Peptostreptococcus evolutus (Prevot) Smith

Summary Peptostreptococcus evolutus was isolated from feces. Its murein containes muramic acid, glucosamine, alanine, d-glutamic acid, lysine and glycine at a molar ratio of about 1:1:3:1:1:1. The ratio of l-alanine: d-alanine is 2,15:1. Glutamic acid is present as an amide.By acid partial hydrolysis of the cell walls and subsequent isolation and identification of the peptides the amino acid sequence of the murein was elucidated. The tetrapeptide is identical with that of most bacteria (l-Ala-d-Glu-NH₂-l-Lys-d-Ala). The crosslinking of the murein is performed by the peptide glycyl-l-alanine. l-alanine is attached to the -amino group of lysine while the amino group of glycine is bound to the carboxyl group of the c-terminal d-alanine of an adjacent tetrapeptide. About 35% glycine and 6% lysine of the murein are dinitrophenylisable indicating that maximally 60% of the possible cross-linkages are realized.

     

Die Aminosäuresequenz eines glycinhaltigen Mureins einiger Stämme von Lactobacillus bifidus

Zusammenfassung Das Murein (Peptidoglycan) von 6 Stämmen Lactobacillus bifidus, die aus der Faeces von Brustkindern oder aus dem Darminhalt von Bienen isoliert worden waren, wies folgendes Molverhältnis auf (auf- bzw. abgerundete Zahlen): Mur:GlcNH₂:Ala:Glu:Lys:Gly=1:1:2:1:1:1. Das Verhältnis l-Ala: d-Ala=1,1:1. Glutaminsäure liegt als Amid vor.Durch die Analyse der Peptide des Partialhydrolysats konnte folgende Aminosäuresequenz erschlossen werden: Das Tetrapeptid besitzt wie bei den meisten Bakterien die übliche Sequenz l-Ala-d-Glu-l-Lys-d-Ala. Glycin ist einerseits an die -Aminogruppe des Lysins, andererseits an die Carboxylgruppe des C-terminalen d-Alanins gebunden und stellt somit die Quervernetzung des Mureins her. Die Dinitrophenylierung der Zellwand ergab, daß rund 50% des Glycins und einige Prozent des Lysins eine freie Aminogruppe tragen. Die Quervernetzung ist demnach nur zu rund 50% durchgeführt.

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The amino acid sequence of the glycine containing murein of some strains of Lactobacillus bifidus

Summary The murein (peptidoglycan) of 6 strains of L. bifidus, isolated from faeces of breast fed infants or from the intestine of bees, respectively, contained muramic acid, glucosamine, alanine, glutamic acid, lysine and glycine at a molar ratio of 1:1:2:1:1:1. The ratio of l-alanine: d-alanine is 1,1: 1. The analysis of the peptides obtained by acid partial hydrolysis indicated that the amino acid sequence of the tetrapeptide is identical with that of most bacteria (l-Ala-d-Glu-l-Lys-d-Ala). Glutamic acid is present as an amide.Glycine is involved in the crosslinking of adjacent muropeptides by forming a bridge between the -aminogroup of lysine and the carboxyl group of a C-terminal d-alanine. About 50% of the glycine is N-terminal, indicating that only 50% of the possible cross linkages are realized.The murein of these strains of L. bifidus resembles the murein of staphylococci, but differs by the number of glycine molecules. While a pentameric glycylpeptide occurs in the murein of staphylococci, only one molecule of glycine is involved in the crosslinkage of the murein described here.

     

Die Aminosäuresequenz des Threonin und Serin enthaltenden Mureins von Bifidobacterium longum Reuter

Zusammenfassung Von 18 Stämmen von Bifidobacterium longum Reuter und einem Stamm von B. lactentis Reuter wurden die Zellwände in üblicher Weise hergestellt. Sie enthielten Mur, GlcNH₂, d-Glu, Ala, l-Orn (l-Lys), Thr, Ser in einem Molverhältnis von rund 1:1:1:4:1:1:1. Die Molverhältnisse änderten sich nicht, wenn die Zellwände mit Trichloressigsäure oder heißem Formamid extrahiert wurden. In einigen Stämmen trat mehr als 1 Mol Glutaminsäure pro Mol Diaminosäure auf. Die zusätzliche Glutaminsäure hatte die l-Konfiguration. Sie war kein Bestandteil des Mureins, sondern einer lysozymunempfindlichen, unbekannten Zellwandkomponente, vermutlich einer Polyglutaminsäure. l-Ornithin war in den meisten Stämmen die dominierende Diaminosäure, während l-Lysin nur mit einem Anteil von 10–20% vertreten war. In 2 Stämmen war l-Lysin dominierend (90%). Die Aminosäuresequenz wurde durch die Analyse der Oligopeptide aus Partialhydrolysaten bestimmt. Die an Mureinsäure gebundenen Peptiduntereinheiten hatten die auch von anderen Mureinen bekannte Sequenz: l-Ala-d-Glu-l-Orn (oder l-Lys)-d-Ala. Glutaminsäure ist wahrscheinlich amidiert, wie aus dem Auftreten von rund 1 Mol NH₃ im Hydrolysat der Zellwände zu schließen ist. Die Interpeptidbrücke besteht aus dem Peptid l-Ala-Thr-l-Ala-l-Ser. Sie ist mit dem C-terminalen Serin an die -Aminogruppe der Diaminosäure der Peptiduntereinheit gebunden. Die Quervernetzung erfolgt zwischen dem N-terminalen Alanin der Interpeptidbrücke zum C-terminalen d-Alanin einer Peptiduntereinheit. Da 4% des gesamten Alanins und 3% der -Aminogruppe des Ornithins dinitrophenylierbar sind, ist anzunehmen, daß die Quervernetzung nur zu etwa 80% verwirklicht ist.

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The amino acid sequence of the threonine and serine containing murein of Bifidobacterium longum reuter

Summary Cell walls of 18 strains of Bifidobacterium longum Reuter and one strain of B. lactentis Reuter were prepared in the usual way. They contained Mur, GlcNH₂, d-Glu, Ala, l-Orn (l-Lys), Thr, Ser in a molar ratio of about 1:1:1:4:1:1:1. The ratio was not changed when the cell walls were extracted by trichloroacetic acid or hot formamide. In some strains more than 1 mole glutamic acid per mole of diamino acid was present. The additional glutamic acid was of the l-rather than the d-form. It was not a constituent of the murein, but of an unknown lysozyme insensitive cell wall component, probably a polyglutamic acid. l-Ornithine was the dominating diamino acid in most strains but l-lysine was also present in a portion of 10 to 20%. In 2 strains l-lysine was dominating (90%).The amino acid sequence was determined by analysing the oligopeptides arising during partial acid hydrolysis. It was shown that the peptide subunits attached to the muramic acid are the same as those of other mureins: l-Ala-d-Glu-l-Orn (or l-Lys)-d-Ala. glutamic acid is probably amidated, since about 1 mole of NH₃ is released by acid hydrolysis of the cell walls. The interpeptide bridge consists of the peptide l-Ala-Thr-l-Ala-l-Ser which is bound by its C-terminal serine to the -amino group of the diamino acid of one peptide subunit and by its N-terminal l-alanine to the C-terminal d-alanine of another peptide subunit. About 4% of the total alanine and 3% of the -amino groups of ornithine of the cell wall can be dinitrophenylated. This indicates that about 20% of the peptide subunits are not crosslinked.

     

Die Aminosäuresequenz von 2,4-Diaminobuttersäure enthaltenden Mureinen bei verschiedenen coryneformen Bakterien und Agromyces ramosus

Zusammenfassung Bie 17 Stämmen von coryneformen Organismen wurde 2,4-Diaminobuttersäure als Bestandteil des Mureins gefunden. In 15 Fällen ergab die genauere Analyse die gleiche Aminosäuresequenz, wie sie schon früher von Perkins (1968) bei Corynebacterium insidiosum beschrieben wurde. In diesem Falle ist die L-2,4-Diaminobuttersäure ein Bestandteil der Peptiduntereinheit, während die D-2,4-Diaminobuttersäure die Quervernetzung zwischen dem Glutaminsäurerest und dem C-terminalen Alanin zweier benachbarter Peptiduntereinheiten herstellt. Das Murein gehört demnach zur Gruppe B nach Schleifer u. Kandler (1972). Die -Aminogruppe der L-2,4-Diaminobuttersäure ist in einigen Fällen acetyliert, in anderen Fällen ist sie frei.Das Murein der beiden anderen Stämme unterscheidet sich in seiner Primärstruktur dadurch, daß nur L-2,4-Diaminobuttersäure vorkommt. Im Falle von C. bovis ist wie bei einigen coryneformen pflanzenpathogenen Stämmen die Diaminosäure der Peptiduntereinheit durch Homoserin ersetzt und die Quervernetzung erfolgt durch das Dipeptid -Gly-L-Dab zwischen Glutaminsäure und D-Alanin. Dieses Murein gehört demnach ebenfalls zur Gruppe B. Dagegen ist das Murein von Arthrobacter sp. Ar 22 eine neue Variante der Gruppe A. Die L-2,4-Diaminobuttersäure ist hier ein Glied der Peptiduntereinheit und die Quervernetzung zwischen der -Aminogruppe der 2,4-Diaminobuttersäure und dem D-Alaninrest einer benachbarten Peptiduntereinheit wird durch das Pentapeptid -L-Asp-L-Ala-Gly-L-Ala-L-Ala gebildet. Außerdem ist die Position 1 der Peptiduntereinheit nicht mit L-Alanin, sondern mit Glycin besetzt. Letzteres ist bisher nur bei Mureinen der Gruppe B, aber nicht bei denen der Gruppe A gefunden worden. Ebenfalls neu ist das Vorkommen von L-Asparaginsäure anstelle der bisher gefundenen D-Form.

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The amino acid sequence of 2,4-diaminobutyric acid containing mureins of various coryneform bacteria and Agromyces ramosus

Summary In 17 strains of coryneform bacteria, 2,4-diaminobutyric acid was found to be a component of the murein (peptidoglycan). A detailed analysis showed that 15 strains contain a murein with the same amino acid sequence as that found in Corynebacterium insidiosum by Perkins (1968). In this case the L-2,4-diaminobutyric acid is a component of the peptite subunit while the D-2,4-diaminobutyric acid serves as interpetide bridge between D-glutamatic and the C-terminal D-alanine residue. Therefore this murein belongs to group B according to Schleifer and Kandler (1972). The -amino group of L-2,4-diaminobutyric acid is in some species acetylated, in others free.The murein of the remaining two strains differs by the lack of D-2,4-diaminobutyric acid. Only L-2,4-diaminobutyric acid is found. In the case of C. bovis, the diamino acid of the peptide subunit is replaced by L-homoserine as found in various plant pathogenic coryneform bacteria. The interpeptide bridge consists of the dipeptide -Gly-2,4-Dab. It connects the D-glutamic acid of one peptide subunit with the C-terminal D-alanine residue of an adjacent peptide subunit. Therefore this murein belongs also to group B.The murein of Arthrobacter sp. Ar 22 is a new varition of group A, however. Here the L-2,4-diaminobutyric acid is a component of the peptide subunit. The interpeptide bridge consists of the pentapeptide -L-Asp-L-Ala-Gly-L-Ala-L-Ala. It connects the -amino group of L-2,4-diaminobutyric acid and the C-terminal D-alanine residue of two peptide subunits. Position 1 of the peptide subunit is occupied by glycine instead of L-alanine as found in all the other mureins of group A so far. Another new feature of this murein is the occurrence of the L-form instead of the D-form of aspartic acid.

     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Die Aminosäuresequenz des Mureins von Lactobacillus coprophilus

Zusammenfassung Das Murein (Peptidoglycan) von Lactobacillus coprophilus enthält Muraminsäure, Glucosamin, Glutaminsäure, Lysin und Alanin in einem molaren Verhältnis von etwa 1:1:1:1:3,3. Das Verhältnis d-Ala: l-Ala beträgt 1:3. Die Aminosäuresequenz der Muropeptide ist wie bei den meisten bisher untersuchten Bakterien l-Ala, d-Glu, l-Lys-d-Ala. Die Glutaminsäure liegt dabei als Amid vor.Die Querverbindung erfolgt durch das Dipeptid l-Alanyl-l-Alanin, das einerseits an die -Aminogruppe des Lysins, andererseits an die Carboxylgruppe des d-Alanins eines benachbarten Muropeptides gebunden ist. Im fertigen Murein sind rund 4% der -Aminogruppen des Lysins und 7,5% der Aminogruppe des Alanins, das sind 30% des N-terminalen l-Alanins der Brückenpeptide, frei. Die Quervernetzung ist demnach nur zu rund 70% verwirklicht.

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The amino acid sequence of the murein of Lactobacillus coprophilus

Summary The murein (peptidoglycan) of Lactobacillus coprophilus contains muramic acid, glucosamine, glutamic acid, lysine and alanine in a molar ratio of about 1:1:1:1:3.3. The ratio of d-/l-alanine is 1:3.1. The amino acid sequence of the muropeptides is identical with that of most bacteria (l-Ala-d-Glu-l-Lys-d-Ala). Glutamic acid is present as an amide.The dipeptide l-alanyl-l-alanine is involved in the cross linking of adjacent muropeptides, by forming a bridge between the -amino group of lysine and the carboxyl group of a C-terminal d-alanine.About 4% of the -amino groups of lysine and 7.5% of the amino groups of alanine (i.e. 30% of the N-terminal l-alanine of the crosslinking-peptides) are free. This indicates, that only 70% of the possible cross-linkages are realized.

     

Die Beeinflussung der Aminosäuresequenz des serinhaltigen Mureins von Staphylococcus epidermidis Stamm 24 durch die Nährbodenzusammensetzung

Zusammenfassung Beim Wachstum von S. epidermidis, Stamm 24, in Hefe-Dextrose-Bouillon weist das Murein folgende Molverhältnisse auf (auf- bzw. abgerundete Zahlen): Mur-GlcNH₂:Ala:Glu:Lys:Gly:Ser=1:1:2,4:1:1:4,2:0,6. Die Glutaminsäure ist amidiert.Durch Isolierung und Identifizierung der Peptide des Partialhydrolysates des Mureins wurde die Aminosäuresequenz bestimmt. Die an die Muraminsäure gebundene Peptiduntereinheit (l-Ala-d-GluNH₂ -l-Lys-d-Ala) stimmt mit der von S. aureus, Copenhagen bzw. S. epidermidis, Stamm 66, überein. Bei knapp einem Drittel der Peptiduntereinheiten ist das C-terminale d-Alanin der Mureinvorstufe nicht abgespalten, so daß diese noch als Pentapeptide vorliegen. Dies konnte aus dem Verhältnis l-Ala/d-Ala (1:1,3), dem Ergebnis der Hydrazinolyse und der Isolierung der Muropeptide nach Spaltung der Zellwände mit Lysozym geschlossen werden.Bei etwa der Hälfte aller aus 5 Glycinresten aufgebauten Interpeptidketten ist ein Glycinrest durch l-Serin ersetzt. Die genaue Position des Serins konnte nicht bestimmt werden. Serin ist sicher nicht direkt an die -Aminogruppe des Lysins gebunden.In selteneren Fällen kann Lysin mit l-Alanin substituiert sein, das N-terminal vorliegt und nicht der Quervernetzung dient.Die Dinitrophenylierung des Mureins ergab, daß in etwa 3,5% der Fälle die Interpeptidketten fehlen und rund ein Drittel der Interpeptidketten nicht quervernetzt ist.Bei Wachstum in einem halbsynthetischen, glycinarmen Medium (Minimal, medium) nimmt der Glycinanteil des Mureins um rund 40% ab, während l-Alanin zunimmt. Es konnte gezeigt werden, daß rund 15% des Mureins ein an die -Amino-gruppe des Lysins gebundenes l-Alanin enthalten, das aber im Unterschied zu S. epidermidis, Stamm 66, nicht mit Glycin substituiert ist, sondern N-terminal bleibt und nicht zur Quervernetzung benützt werden kann. Weiterhin liegen hier rund 35% des Lysins unsubstituiert vor, und nur etwa 50% der Peptiduntereinheiten weisen eine Pentaglycyl-Interpeptidkette auf. Die Quervernetzung des Mureins ist bei den in Minimalmedium gewachsenen Zellen nur zu rund 30% durchgeführt. Bei Zusatz von Glycin zum Minimal-Nährboden wird der Glycingehalt im Murein erhöht, während der extra Alaninanteil praktisch verschwindet. Serinzusatz erhöht nicht nur den Serin-, sondern auch den Glycinanteil. Bei Alaninzusatz dagegen wird der Alaningehalt im Murein etwas erhöht und der Glycingehalt weiter erniedrigt.Die Ergebnisse dieser Untersuchungen wurden mit entsprechenden, vorläufigen Versuchen bei S. epidermidis, Stamm 66 und S. aureus, Stamm Copenhagen, verglichen. Es zeigt sich, daß trotz starker modifikativer Veränderungen der Mureinzusammensetzung eindeutige genetische Unterschiede zwischen diesen drei Stämmen vorliegen.

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The effect of nutrition on the amino acid sequence of the serine containing murein of Staphylococcus epidermis strain 24

Summary The murein (peptidoglycan) of S. epidermidis strain 24 contains Mur GlcNH₂, Ala, Glu, Lys, Gly, Ser at a molar ratio of about 1:1:2.4:1:1:4.2:0.6 when grown in a yeast extract dextrose medium. Glutamic acid occurs as an amide.The amino acid sequence was determined by analysing the oligopeptides from partial acid hydrolysate. The tetrapeptide bound to the muramic acid (l-Ala-d-Glu-NH₂-l-Lys-d-Ala) is identical with those found in S. aureus and S. epidermidis strain 66. About 1/3 of the muropeptides is still present as pentapeptides, since the second d-alanine of the muramyl pentapeptide precursor is not split off. This fact is indicated by the ratio of l-Ala/d-Ala of 1:1.3, the isolation of muropentapeptides from the lysozyme lysates and by the result of the hydrazinolysis.About 50% of the pentaglycine interpeptide chains contain one mole of l-serine. The exact position of l-serine could not be determined. However, it could be shown, that serine is never bound to the -amino group of lysine. In very rare cases, the -amino group of lysine is substituted by l-alanine which remains N-terminal and can not be used for crosslinkages.As shown by dinitrophenylation, about 3.5% of the -amino groups of lysine is free and about 50% of the interpeptide chains are not cross-linked.If the organism is grown in a glycine deficient minimal medium, the glycine content of the murein drops by 40%, while l-alanine increases. Here, about 15% of the -amino groups of lysine is substituted by l-alanine, which again is not used for cross-linkages. Another 35% of the -amino groups of lysine remain free. From the existing interpeptide chains 30% are not cross-linked.The addition of glycine to the minimal medium causes an increase of the glycine content in the murein, however, the extra alanine protion nearly disappears. The addition of serine leads to an increase of not only the serine portion but also the glycine portion in the murein. However, when alanine is added the alanine portion of murein is slightly increased and the glycine portion further decreased.The results of these experiments were compared to corresponding preliminary experiments with S. epidermidis (strain 66) and S. aureus (strain Copenhagen). In spite of modificative changes in the murein composition, clear genetical differences between the 3 strains were obvious.

     

Peptidoglycan types and cytochrome patterns of strains of Oerskovia turbata and O. xanthineolytica

Determination of the primary structure of the peptidoglycan of 15 strains of Oerskovia showed that three different peptidoglycan types occur. Oerskovia xanthineolytica strains contain the l-Lys-d-Ser--d-Asp type, whereas Oerskovia turbata strains show the new peptidoglycan types l-Lys-l-Thr--d-Asp or l-Lys-l-Thr--d-Glu, respectively. Research on the cytochromes of Oerskovia revealed the presence of a, b and c types. O. turbata can be clearly distinguished from O. xanthineolytica by the occurrence of cytochrome a ₁ in cells, isolated from the stationary phase. The following conclusions were made: O. turbata and O. xanthineolytica can be clearly separated on the basis of different peptidoglycan types and cytochrome patterns. This distinction is in perfect correlation with the classical separation method of O. turbata and O. xanthineolytica on the basis of xanthine degradation. l-Lys-d-Ser--d-Asp peptidoglycan type does not only occur in O. xanthineolytica but also in some coryneform bacteria such as Corynebacterium manihot (Fiedler et al. 1970), Cellulomonas cartae (Stackebrandt et al. 1978; Stackebrandt and Kandler 1980), Brevibacterium fermentans and Nocardia cellulans.This paper is respectively dedicated to Professor Dr. O. Kandler, on the occasion of his 60th birthday     

Purification,characterization and regulation of a monomeric l-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and -ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 mol min^-1 mg^-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPT hexulose-6-phosphate isomerase - FPLC fast protein liquid chromatography     

Glycosylation with thioglycosides activated by dimethyl(methylthio)sulfonium tetrafluoroborate: synthesis of two trisaccharide glycosides corresponding to the blood group A and B determinants

Two trisaccharide glycosides,p-trifluoroacetamidophenylethyl 3-O-(2-acetamido-2-deoxy--d-galactopyranosyl)-2-O-(-l-fucopyranosyl)--d-galactopyranoside andp-trifluoroa-cetamidophenylethyl 2-O-(-l-fucopyranosyl)-3-O-(-d-galactopyranosyl)--d-galactopyranoside, corresponding to the human blood group A and B determinants, were synthesized. A key fucosylgalactosyl disaccharide derivative was glycosylated with galactosaminyl or galactosyl donors, respectively. Dimethyl (thiomethyl)sulfonium tetrafluoroborate was used for thioglycoside activation in coupling reactions.     

Structure-activity relationship of a novel peptide substrate for p60c-src protein tyrosine kinase

Summary We recently reported the identification of a peptide (YIYGSFK) as an efficient substrate for p60^c-src using a random combinatorial peptide library screening method. Over 70 analogues of YIYGSFK were designed and synthesized on beads and their phosphorylation on solid phase by p60^c-src was quantitated by the PhosphorImager. A hydrophobic l-amino acid in position 2 and a basic amino acid in position 7 proved crucial for activity as a substrate. In addition, the l-tyrosine residue at position 3 was critical as the phosphorylation site and was found to be stereospecific, as substitution with the d-enantiomer at this position rendered the peptide totally inactive.Abbreviations -alanine - -aminocaproic acid - Ac N-acetyl - BOP benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - Cha l-cyclohexylalanine - Chg l-cyclohexylglycine - Dab l-diaminobutyric acid - Dap l-diaminoproprionic acid - DIEA N,N-diisopropylethylamine - DMF dimethylformamide - Fmoc fluorenylmethyloxycarbonyl - HOBt N-hydroxybenzotriazole - MeF N-methyl-l-phenylalanine - MeG N-methylglycine - MeI N-methyl-l-isoleucine - MES 2-[N-morpholinolethanesulfonic acid - Nle l-norleucine - Orn l-ornithine - TFA trifluoroacetic acid - Z-Sar benzyloxycarbonyl-sarcosine - Z-Tyr benzyloxycarbonyl-l-tyrosine     

Induction of extracellular arabinases on monomeric substrates in Aspergillus niger

The induction of extracellular arabinases by pentose sugars and polyols generated by the metabolic pathway of l-arabinose and d-xylose catabolism in Aspergillus niger was investigated. Induction occurred with l-arabinose and l-arabitol but not with d-xylose or xylitol. l-arabitol in particular was found to be a good inducer for -l-arabinofuranosidase and endo-arabinase activities. Western blotting analysis showed both -l-arabinofuranosidase A and B to be present. No induction was observed using d-arabitol. Unlike the wild type A. niger N402 strain, the A. niger xylulose kinase negative mutant N572 also showed induction of -l-arabinofuranosidases A and B and endo-arabinase activity on d-xylose and xylitol. This is due to metabolic conversion of these compounds leading to the accumulation of both xylitol and l-arabitol in this mutant, the latter of which then acts as inducer. The induction of the two -l-arabinofuranosidases and endo-arabinase is under the control of two regulatory systems namely pathway specific induction and carbon catabolite repression. Under derepressing conditions in the wild type only -l-arabinofuranosidase B could be detected by Western blotting analysis. This indicates that -l-arabinofuranosidase B is of importance in the initiation of specific induction of the various arabinose activities in A. niger grown on arabinose containing structural polysaccharides.Abbreviations PNA p-nitrophenyl--l-arabinofuranoside     

Cloning,characterization, and mutational analysis of a highly active and stable <Emphasis Type="SmallCaps">l</Emphasis>-arabinitol 4-dehydrogenase from <Emphasis Type="Italic">Neurospora crassa</Emphasis>

An NAD⁺-dependent l-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer and contained two Zn²⁺ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (SDHs). High enzymatic activity was observed for substrates l-arabinitol, adonitol, and xylitol and no activity for d-mannitol, d-arabinitol, or d-sorbitol. The enzyme showed strong preference for NAD⁺ but also displayed a very low yet detectable activity with NADP⁺. Mutational analysis of residue F59, the single different substrate-binding residue between LADs and d-SDHs, failed to confer the enzyme the ability to accept d-sorbitol as a substrate, suggesting that the amino acids flanking the active site cleft may be responsible for the different activity and affinity patterns between LADs and SDHs. This enzyme should be useful for in vivo and in vitro production of xylitol and ethanol from l-arabinose. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Glycosidations with thioglycosides activated by sulfuryl chloride/trifluoromethanesulfonic acid: synthesis of a human blood group B trisaccharide glycoside

The trisaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-O-(-l-fucopyranosyl)-3-O-(-d-galactopyranosyl)--d-galactopyranoside, corresponding to the human blood group B determinant, was synthesized. Thioglycosides activated by sulfuryl chloride/trifluoromethanesulfonic acid were used as glycosyl donors in the construction of the three glycosidic linkages.     

Lipopolysaccharide of Providencia rettgeri

The chemical constitutional analysis of the lipopolysaccharide (LPS) isolated from Providencia rettgeri was carried out. Polyacrylamide gel electrophoresis using sodium dodecylsulfate or sodium deoxycholate showed that the lipopolysaccharide mostly consisted of short sugar chains. The lipid A was precipitated out after mild acid hydrolysis of LPS. From the supernatant degraded polysaccharide and unsubstituted core fractions were isolated. Compositional analysis of the core material revealed the presence of galacturonic acid, galactose, glucose, glucosamine, l-glycero-d-manno-heptose, 3-deoxy-d-manno-octulosonic acid, alanine and phosphorus. Methylation analysis of the core material indicated the presence of terminal units of glucose, galacturonic acid and glucosamine. The chemical structure of the lipid A was elucidated. It constitutes a -1,6-glucosamine disaccharide substituted on either side by ester and glycosidically-bond phosphate residues. The ester-bound phosphate was found to be substituted by a 4-amino-4-deoxy-l-arabinosyl residue. The amino groups of the backbone disaccharide are N-acylated by 3-O-(14:0)14:0 and 3-O-14:0.Two hydroxyl groups of the disaccharide are esterified by 3-O-(14:0)14:0 and 3-O-14:0. The taxonomical importance of these structural details will be discussed.Abbreviations LPS lipopolysaccharide - l-d-heptose l-glycero-d-manno-heptose - dOclA 3-deoxy-d-manno-octulosonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - PS degraded polysaccharide - glc-ms combined gas liquid chromatography-mass spectrometry     

Biosynthesis of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane by methanogenic bacteria

In an attempt to establish the nature of the ammonium-assimilation products which mediate the inhibition by ammonium of nitrate uptake in cyanobacteria, the effect of different amino acids on nitrate utilization by intact Anacystis nidulans cells has been assayed. To exclude an indirect inhibition of nitrate uptake through the ammonium which the amino acids might release, the cells were pretreated with l-methionine-d,l-sulfoximine (MSX), a potent inactivator of glutamine synthetase. Under these conditions, several l-amino acids, but not the corresponding d-isomers, affected nitrate utilization to a variable extent, causing inhibitions ranging between 20 and 80% when added at 20 mM concentration.For most of the inhibitory amino acids, including l-isoleucine, l-leucine and l-valine, a correlation was found between their ability to act as amino group donors to -ketoglutarate, in reactions catalyzed by A. nidulans cell-free extracts, and their inhibitory effect on nitrate utilization. l-Glutamine, l-asparagine and glycine, being effective inhibitors of nitrate utilization, were poor substrates for the transaminating activity to -ketoglutarate, however. The possible role of the latter amino acids as mediators in the ammonium-promoted inhibition of nitrate uptake is discussed.Abbreviations MSX l-methionine-d,l-sulfoximine - MTA-5 mixed alkyltrimethylammonium bromide - Mops morpholinopropane sulfonic acid     

Verwertung von Trimethylammoniumverbindungen durch Acinetobacter calcoaceticus

Zusammenfassung Die Verwertung von Carnitin und Carnitinderivaten (O-Acylcarnitine, Carnitincarboxyl-derivate) und strukturverwandten Trimethylammoniumverbindungen (Betaine und Stickstoffbasen) durch Acinetobacter calcoaceticus wurde anhand des Wachstums und des quantitativen Nachweises der Metabolite untersucht. Der Stamm wuchs auf l-Carnitin, l-O-Acylcarnitinen und -Butyrobetain als jeweils einziger C-Quelle. Der Verbrauch dieser Verbindungen und das Wachstum korrelierten mit der Spaltung der C-N-Bindung und mit dem gebildeten Trimethylamin. d-Carnitin wurde metabolisiert, wenn als zusätzliche C-Quelle l-Carnitin im Nährmedium vorhanden war, oder wenn die Bakterien mit l-oder dl-Carnitin vorinkubiert worden waren. Mit d-Carnitin als einziger C-Quelle wuchsen die Bakterien jedoch nicht. Die Bakterien oxidierten Cholin zu Glycinbetain in Gegenwart einer zusätzlichen C-Quelle, Glycinbetain selbst wurde nicht assimiliert. In Hinsicht auf den Abbau quaternärer Stickstoffverbindungen besitzt Acinetobacter calcoaceticus im Vergleich zu anderen Carnitin-verwertenden Bakterienarten einen für ihn charakteristischen Stoffwechselweg.

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Utilization of trimethylammonium-compounds by Acinetobacter calcoaceticus

The utilization of carnitine and carnitine derivatives (O-acylcarnitines, carnitine carboxylderivatives) and structure-related trimethylammonium-compounds (betaines and nitrogen-bases) by Acinetobacter calcoaceticus was studied by means of the control of growth and the quantitative detection of metabolites. The strain grew only on l-carnitine, l-O-acylcarnitines, and -butyrobetaine as the sole carbon sources. The utilization of these compounds and the growth correlated with the cleavage of the C-N bond and thereby with the formation of trimethylamine. d-Carnitine was metabolized, if an additional carbon source, like l-carnitine, was present in the incubation mixture, or if the bacteria were preincubated with l-or dl-carnitine, but no growth was observed on d-carnitine as the sole carbon source. The bacteria oxidized choline to glycinebetaine in the presence of additional carbon sources, glycinebetaine itself was not assimilated. With regard to the catabolism of quaternary nitrogen compounds Acinetobacter calcoaceticus shows a different pathway in comparison with other bacterial species metabolizing carnitine.

     

The xylogalactan sulfate from Chondria macrocarpa (Ceramiales,Rhodophyta)

A structure is proposed for the complex xylogalactan sulfate from Chondria macrocarpa. The hot-water extract of C. macrocarpa was desulfated or alkali-treated and Smith degraded. Constituent sugars and their substitution patterns were identified using a modified Hakamori methylation procedure suited to sulfated polysaccharides and a double hydrolysis-reduction protocol that yielded derivatives from all of the sugar residues, including the labile 3,6-anhydrogalactosyl residues. The polymer has an agar-type backbone of alternating 3-linked \-d- and 4-linked -L-galactopyranosyl units. The d-residues are partially sulfated on O-2 (50%) and O-6 (20–30%). About 40% of the l-residues are present as the 3,6-anhydride and 25% as its precursor l-galactose 6-sulfate. A significant proportion of the remaining l-galactosyl residues have both a d-xylopyranosyl substituent on O-3 and a sulfate ester on O-6 and are stable to alkali.