d‐Glucose‐6‐Phosphate Dehydrogenase (Entner‐Doudoroff Enzyme) from Pseudomonas fluorescens

Abstract

1. The existence of two different ~-glucose-6-phosphate dehydrogenases in Pseudomonas Juorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway.2. A procedure is described for the isolation of that ~-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44 %. The final preparation, having a specific activity of about 300 pmol NADH formed per min per mg protein, was shown to be homogeneous.3. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000.4. The p l of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9.5. The Entner-Doudoroff enzyme showed specificity for NAD' as well as for NADP' and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory.6. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of P-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase.In organisms of the genus Pseudomonas glucose 6-phosphate may be metabolized via the Entner-Doudoroff pathway, which is characterized by the dehydratation of 6-phospho-~-gluconate to 2-keto-3-deoxy-6- phospho-D-gluconate and its subsequent cleavage to pyruvate and ~-glyceraldehyde-3-phosphate [ 1,2]. Although it is the sole pathway of D-glucose 6-phosphate metabolism in Ps. saccharophila, the additional existence of the hexose monophosphate pathway has been demonstrated in other species of Pseudomonads [3 -51. Both these pathways have in common the initial dehydrogenation of D-glucose 6-phosphate at carbon-1 and the subsequent hydrolysis of 6-phospho-~-glucono-l,5-lactone. According to that, the overlapping catabolic Entner-Doudoroff and anabolic hexose monophosphate pathways must be susceptible to metabolic regulation. The activity of ~-glucose-6-phosphate dehydrogenase from a number of Pseudomonads has been demonstrated to be subject to metabolic control by ATP or related triphosphates [6-91. Furthermore in the case of Ps. multivorans two ~-glucose-6-phosphate dehydrogenases have been

Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production

“…These data indicate that Zwf-2 is active even when the energy status of the cell is good, probably taking part in the pentose phosphate pathway operating in the anabolic direction. Lessmann et al (5) reported that the most highly expressed isoenzyme, now annotated as Zwf-1, showed a slight preference for NAD ϩ (5), while our results indicate that both enzymes prefer NADP ϩ to NAD ϩ as a cofactor. The reasons for this discrepancy are not known but could, for instance, be due to strain-dependent differences.…”

“…We wanted to study the contributions of glucose dehydrogenase (Gcd) and glucose-6-phosphate dehydrogenase (G6PDH; also called Zwf) to these processes. Lessmann et al (5) have described two different G6PDHs from P. fluorescens and have shown that the activity of one of these isoenzymes did not vary much with the carbon sources tested, while the other was induced by glucose (5). It is not clear, however, whether the two P. fluorescens Zwf enzymes have strictly defined roles, one being catabolic and the other anabolic, or if either one would be sufficient for growth.…”

The Pseudomonas aeruginosa devB/SOL Homolog, pgl , Is a Member of the hex Regulon and Encodes 6-Phosphogluconolactonase

“…U14553). While P. fluorescens and Pseudomonas cepacia (reclassified as Burkholderia cepacia) have two glucose-6-phosphate dehydrogenases that function with either NAD ϩ (in a catabolic role) or NADP ϩ (in an anabolic role) (7,20,25), it is clear that P. aeruginosa has a single glucose-6-phosphate dehydrogenase activity (Zwf) capable of using either NAD ϩ or NADP ϩ (22,28). One interpretation consistent with a single zwf gene is that the open reading frame with homology to devB/SOL represents a 6-phosphogluconolactonase.…”

Transient expression and flux changes during a shift from high to low riboflavin production in continuous cultures ofBacillus subtilis

“…Since this major flux change was not accompanied by any change in PP pathway or glycolytic transcript levels, PP pathway activity was not controlled at the transcriptional level as was suggested before (Blenke et al, 2003). Enzyme level control of this flux may be realized through the feedback inhibition of glucose-6-P dehydrogenase by NADPH known in other bacteria (Lessmann et al, 1975;Moritz et al, 2000). In this context, the inverted expression trends of the NADH-dependent, glycolytic GapA and the NADPH-dependent, gluconeogenic GapB isoenzymes (Fillinger et al, 2000) may be relevant.…”