Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

Abstract

In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Dpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH R46E,Q47E . Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Dpgi E. coli strains were replaced by DNA encoding LmG6PDH R46E,Q47E . Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Dpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.

Growth adaptation ofgndandsdhCB Escherichia colideletion strains diverges from a similar initial perturbation of the transcriptome

“…It has been shown previously that disrupting the gnd gene resulted in non appreciable changes to growth rate, but induces major changes in fluxes through the PPP and TCA cycle [1][2][3] . Similar observations have also been made of zwf mutants 4,5 , which demonstrate that metabolic networks can rapidly adjust enzyme level and flux without an appreciable cost to growth rate.…”

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

“…Because the genes gnd and zwf-2 are in the same operon, the activity of 6phosphogluconate dehydrogenase (GND) could indirectly indicate the expression of the protein encoded by zwf-2, annotated as another G6PDH. 13 C-labeling experiments had shown that the oxidative branch of the pentose-phosphate pathway is operative in P. putida KT2440 when glucose is the sole carbon source [2], so we decided to measure the activity of GND in the crude cellular extracts ( Table 3). The registered GND activities together with the biased estimations of E (Fig.…”

Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation, Kinetics and Evolutionary Studies

“…However, this analysis involved considerably shorter simulations (0.5 ps), not intended to evaluate the stability of protein-ligand binding. In addition, we previously reported MD simulations of the dual Lm G6PDH with NAD + and NADP + , based on its crystallographic complexes [ 11 ]. In this enzyme, the R46 residue is homologous to R50 of Ec G6PDH ( Fig 6 ) and has been shown to perform an equivalent function in the interaction with the 2’-phosphate of NADP + [ 8 ].…”

“…coli enzyme and is important for interacting with the 2’-phosphate and the ribose-3’-hydroxyl group of NADP + . Interestingly, in opposition to Ec G6PDH, the simulations of the Lm G6PDH-NAD + complex showed that the ribose-adenine region was stable in its pocket, mainly because of stable hydrogen bonding with the main chain atoms of T14 and R46, together with a stabilizing interaction with the side chains of Q47 and T14 [ 11 ].…”

“…Particularly, complexes of Lm G6PDH with NADP + (PDB ID 1H9A), NAD + (PDB ID 1H94), NADPH and D-glucose (PDB ID 1E7Y) have provided information about the residues modulating the preference for both dinucleotides, which have also been evaluated by site-directed mutations. We recently published the effect of the double mutant R46E-Q47E on the cofactor preference of Lm G6PDH [ 11 ]. In fact, we demonstrated that the mutant performed as a specific NADH-producer in vivo .…”