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Der Biosyntheseweg der RNS-Ribose in Hydrogenomonas eutropha Stamm H 16 und Pseudomonas facilis

The biosynthetic pathway of RNA ribose in Hydrogenomonas eutropha Strain H 16 and Pseudomonas facilis

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Summary

The enzymes involved in the degradation of fructose and gluconate via the Entner-Doudoroff-pathway as well as those involved in the oxidative pentose phosphate pathway have been determined in crude extracts of Hydrogenomonas eutropha strain H 16 and of Pseudomonas facilis after either autotrophic growth or heterotrophic growth on fructose or gluconate as substrates. In H. eutropha fructose induces all enzymes of the Entner-Doudoroff-pathway, gluconate induces only glucokinase, 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase. In contrast, in P. facilis both substrates induce the entire set of enzymes. The absence of 6-phosphogluconate dehydrogenase in H. eutropha and the presence of a NAD-linked 6-phosphogluconate dehydrogenase in P. facilis have been confirmed. Otherwise, the enzyme activities in fully induced fructose grown cells of both species are similar.

Incorporation experiments were performed using both bacterial species and employing U-14C-, 1-14C-, and 6-14C-fructose as well as 1-14C- and 6-14C-gluconate as substrates. Ribose was isolated from RNA and fermented by Lactobacillus plantarum with the production of acetic and lactic acids. By stepwise degradation of the acids and by quantitative measurement and scintillation counting of the carbon dioxide formed the specific radioactivity of each carbon atom has been determined.

The results demonstrate that in strain H 16 ribose is formed exclusively via the non-oxidative reactions of the pentose phosphate pathway. Carbon atoms 1 to 3 of gluconate do not significantly contribute to gluconeogenesis.

With P. facilis an almost identical labelling pattern was observed, indicating that the oxidative reactions of the pentose phosphate pathway via 6-phosphogluconate dehydrogenase are quantitatively of minor importance for ribose synthesis than the transaldolase-transketolase reactions.

Zusammenfassung

Die am Fructose- und Gluconatabbau über den Entner-Doudoroff-Weg beteiligten Enzyme sowie die Enzyme des oxydativen Pentosephosphat-Weges wurden in Rohextrakten von Hydrogenomonas eutropha Stamm H 16 und Pseudomonas facilis, sowohl nach autotrophem Wachstum als auch nach heterotrophem Wachstum auf Fructose oder Gluconat, bestimmt. Fructose induziert in H. eutropha alle Enzyme des Entner-Doudoroff-Weges, Gluconat nur die Gluconokinase, die 6-Phosphogluconat-Dehydratase und die 2-Keto-3-desoxy-6-phosphogluconat-Aldolase. Dagegen induzieren in P. facilis beide Substrate den gesamten Enzymsatz. Das Fehlen der 6-Phosphogluconat-Dehydrogenase in H. eutropha und das Vorhandensein einer NAD-abhängigen 6-Phosphogluconat-Dehydrogenase in P. facilis wurden bestätigt. Die Enzymaktivitäten in voll induzierten, auf Fructose gewachsenen Zellen beider Arten sind ähnlich.

Mit beiden Stämmen wurden Einbauexperimente mit U-14C-, 1-14C- und 6-14C-Fructose sowie 1-14C- und 6-14C-Gluconat als Substrate durchgeführt. Die Ribose wurde aus der RNS isoliert und durch Lactobacillus plantarum fermentativ in Essigund Milchsäure gespalten. Die spezifische Radioaktivität der einzelnen C-Atome wurde durch schrittweisen Abbau der Säuren, quantitative Bestimmung des dabei entstehenden 14CO2 und Messung der darin enthaltenen absoluten Radioaktivität ermittelt.

Die Ergebnisse zeigen, daß die Ribose in Stamm H 16 ausschließlich über die nicht-oxydativen Reaktionen des Pentosephosphat-Weges gebildet wird. Die C-Atome 1,2 und 3 des Gluconats tragen nicht signifikant zur Gluconeogenese bei.

Das Markierungsmuster der Ribose aus P. facilis ist mit dem von Stamm H 16 nahezu identisch. Die oxydativen Reaktionen des Pentosephosphat-Weges über die 6-Phosphogluconat-Dehydrogenase sind von quantitativ geringerer Bedeutung als die Transaldolase-Transketolase-Reaktionen.

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Abbreviations

ATP:

Adenosin-5′-triphosphat

DAP:

Dihydroxyacetonphosphat

E-4-P:

Erythrose-4-phosphat

ED:

Entner-Doudoroff

EDTA:

Äthylen-diamin-tetraessigsäure

FDP(ase):

Fructose-1,6-diphosphat(ase)

F-6-P:

Fructose-6-phosphat

G-6-P(-DH):

Glucose-6-phosphat(-Dehydrogenase)

GAP:

Glycerinaldehyd-3-phosphat

GDH:

Glycerin-1-phosphat-Dehydrogenase

GK:

Gluconokinase

HK:

Hexokinase

KDPG:

2-Keto-3-desoxy-6-phosphogluconat

LDH:

Lactat-Dehydrogenase

NAD(H2):

Nicotin-amid-adenin-dinucleotid (reduziert)

NADP(H2):

Nicotinamid-adenin-dinucleotidphosphat (reduziert)

PGI:

Phosphoglucose-Isomerase

PP:

Pentosephosphat

6-PG(-DH):

6-Phosphogluconat(-Dehydrogenase)

6-PG-DHT:

6-Phosphogluconat-Dehydratase

R-5-P:

Ribose-5-phosphat

Ru-5-P:

Ribulose-5-phosphat

Su-7-P:

Seduheptulose-7-phosphat

TA:

Transaldolase

TEA:

Triäthanolaminhydrochlorid

TIM:

Triosephosphat-Isomerase

TK:

Transketolase

TPP:

Thiaminpyrophosphat

Tris:

Tris-(hydroxymethyl)-aminomethan

Xu-5-P:

Xylulose-5-phosphat

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  1. Strahlen- und Umweltforschung mbH., Institut für Mikrobiologie der Gesellschaft für, München, in Göttingen

    B. Bowien & H. G. Schlegel

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  1. B. Bowien
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  2. H. G. Schlegel
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Bowien, B., Schlegel, H.G. Der Biosyntheseweg der RNS-Ribose in Hydrogenomonas eutropha Stamm H 16 und Pseudomonas facilis . Archiv. Mikrobiol. 85, 95–112 (1972). https://doi.org/10.1007/BF00409291

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