Quantifying NAD(P)H production in the upper Entner–Doudoroff pathway from <i>Pseudomonas putida</i> KT2440

Olavarría, Karel; Marone, Marina Püpke; Oliveira, Henrique da Costa; Roncallo, Juan Camilo; Vasconcelos, Fernanda Nogales da Costa; Silva, Luiziana Ferreira da; Gomez, José Gregório Cabrera

doi:10.1016/j.fob.2015.11.002

Cited by 17 publications

(18 citation statements)

References 39 publications

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“…The rapid-equilibrium random ordered mechanism was the best model to explain the results observed in all cases, similarly to previous findings reported for the G6PDH-A isoform of strain KT2440 (19). Importantly, the main differences between G6PDH-A and G6PDH-B were detected in the K M values for NADP + and G6P, which indicate that the response of these enzymes to changes in the cytoplasmic concentrations of these metabolites could be different (Table 1) .…”

Section: Resultssupporting

confidence: 89%

“…Although most G6PDH are described as NADP + -dependent enzymes, previous reports suggested that dual or even NAD + -preferring homologues exist in several bacterial species, including Pseudomonas and close relatives (17–19, 42–44). The kinetic properties of the G6PDH-B and G6PDH-C isoforms present in strain KT2440 have not been studied so far, and whether G6P oxidation catalyzed by these variants would funnel electrons into the NADH or NADPH pools remains unclear.…”

Section: Resultsmentioning

confidence: 99%

“…The dual cofactor specificity of G6PDH-B probably balances redox cofactor production in a narrow window just by demand, as the NADPH/NADH ratio associated to G6PDH activity is highly dependent on substrate (i.e. NADP + and NAD + ) availability (19). An increase in the demand of a specific cofactor leads to a higher concentration of its oxidized form, thereby shifting substrate availability and, subsequently, the cofactor output.…”

Section: Discussionmentioning

confidence: 99%

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Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes inPseudomonas putidareveals a general principle underlying glycolytic strategies in bacteria

Volke

Olavarría

Nikel

2021

Preprint

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Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase also provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity towards nicotinamide adenine dinucleotide phosphate (NADP+), some variants accept nicotinamide NAD+ similarly (or even preferentially). Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from a genomic, genetic and biochemical perspective. P. putida represents an ideal model to tackle this endeavor, as its genome encodes numerous gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities, and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic ‘gatekeepers’ for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. Multiplication of G6PDH isoforms in these species goes hand-in-hand with low NADP+ affinity at least in one G6PDH isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy towards balancing the relative production of NADPH and NADH.ImportanceProtein families have likely arisen during evolution by gene duplication and divergence followed by neo-functionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the co-existence of multiple isozymes in central carbon catabolism remains relatively unexplored. We have adopted the metabolically-versatile soil bacterium Pseudomonas putida KT2440 as a model to interrogate the physiological and evolutionary significance of co-existing glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs encoded in this bacterium display distinct biochemical properties, especially at the level of cofactor preference, impacting bacterial physiology in a carbon source-dependent fashion. Furthermore, the presence of multiple G6PDHs differing in NAD+- or NADP+-specificity in bacterial species strongly correlates with their predominant metabolic lifestyle. Our findings support the notion that multiplication of genes encoding cofactor-dependent dehydrogenases is a general evolutionary strategy towards achieving redox balance according to the growth conditions.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes inPseudomonas putidareveals a general principle underlying glycolytic strategies in bacteria

Volke

Olavarría

Nikel

2021

Preprint

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“…The differences in activity mediated by each carbon source became more evident when analyzing the two major dehydrogenases of the oxidative PP pathway, i.e., glucose‐6‐ P dehydrogenase (Zwf) and gluconate‐6‐ P dehydrogenase (Gnd). These enzymes display a preference for NADP + as the cofactor (Olavarria et al ., ), and their capability to contribute NADPH under biodegradation conditions was clear: the Zwf activity increased three‐ and four‐fold in benzoate and m ‐xylene cultures as compared to that observed on citrate, and the Gnd activity augmented ca. three‐fold for the same comparison.…”

Section: Resultsmentioning

confidence: 95%

“…We thus measured the intracellular concentration of NADPH, as it is the redox cofactor that seemed to be over‐produced under biodegradation conditions. The NADPH concentration in exponentially‐growing KT2440 cells from glucose cultures was 276 ± 28 μM, consistent with previous reports (Chavarría et al ., ; Nikel et al ., ; Olavarria et al ., ). In citrate cultures, this value increased 1.8‐fold, reflecting the gluconeogenic demand of NADPH under these conditions.…”

Section: Resultsmentioning

confidence: 97%

Pyridine nucleotide transhydrogenases enable redox balance of Pseudomonas putida during biodegradation of aromatic compounds

Nikel

Pérez‐Pantoja

Lorenzo

2016

Environmental Microbiology

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The metabolic versatility of the soil bacterium Pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. Biodegradation of aromatics occurs via the pathway encoded in the archetypal TOL plasmid pWW0, yet the effect of running such oxidative route on redox balance against the background metabolism of P. putida remains unexplored. To answer this question, the activity of pyridine nucleotide transhydrogenases (that catalyze the reversible interconversion of NADH and NADPH) was inspected under various physiological and oxidative stress regimes. The genome of P. putida KT2440 encodes a soluble transhydrogenase (SthA) and a membrane-bound, proton-pumping counterpart (PntAB). Mutant strains, lacking sthA and/or pntAB, were subjected to a panoply of genetic, biochemical, phenomic and functional assays in cells grown on customary carbon sources (e.g., citrate) versus difficult-to-degrade aromatic substrates. The results consistently indicated that redox homeostasis is compromised in the transhydrogenases-defective variant, rendering the mutant sensitive to oxidants. This metabolic deficiency was, however, counteracted by an increase in the activity of NADP -dependent dehydrogenases in central carbon metabolism. Taken together, these observations demonstrate that transhydrogenases enable a redox-adjusting mechanism that comes into play when biodegradation reactions are executed to metabolize unusual carbon compounds.

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Host Organism:Pseudomonas putida

Poblete‐Castro

Acuña

Nikel

et al. 2016

Industrial Biotechnology

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Quantifying NAD(P)H production in the upper Entner–Doudoroff pathway from Pseudomonas putida KT2440

Abstract: HighlightsThe first kinetic characterization of PputG6PDH-1 is presented.The relative production of NADH and NADPH by PputG6PDH-1 is quantified.The stoichiometric matrix of in silico metabolic models for Pseudomonas putida must be modified.

Cited by 17 publications

References 39 publications

Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes inPseudomonas putidareveals a general principle underlying glycolytic strategies in bacteria

Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes inPseudomonas putidareveals a general principle underlying glycolytic strategies in bacteria

Pyridine nucleotide transhydrogenases enable redox balance of Pseudomonas putida during biodegradation of aromatic compounds

Host Organism:Pseudomonas putida

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