Cofactor Specificity of Glucose-6-Phosphate Dehydrogenase Isozymes in Pseudomonas putida Reveals a General Principle Underlying Glycolytic Strategies in Bacteria

Abstract

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 provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity toward NADP+, some variants accept 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 genomic, genetic, and biochemical perspectives. P. putida represents an ideal model to tackle this endeavor, as its genome harbors 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. The abundance of G6PDH isoforms in these species goes hand in hand with low NADP+ affinity, at least in one isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy toward balancing the relative production of NADPH and NADH. IMPORTANCE Protein families have likely arisen during evolution by gene duplication and divergence followed by neofunctionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the coexistence 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 coexisting glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs 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 toward achieving redox balance according to the growth conditions.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

“…Among these, 45 were identified to have metabolic functions ( Table S4 ). Within central carbon metabolism, a down-regulation of gluconokinase (GnuK), glucose 6-phosphate 1-dehydrogenase [ZwfA [77]], and 6-phosphogluconolactonase (Pgl) was observed together with an up-regulation of 2-keto-gluconate-6-P (2K6PG) reductase (KguD; Figure S4 ). This pattern suggests a shift from the preferred glucose utilization route [i.e., glucose oxidation and gluconate uptake [29]] towards further oxidation to 2KG and utilization thereof.…”

“…During growth on glucose, 26% of membrane-bound ubiquinol in the electron transport chain is predicted to be produced by direct glucose oxidation (as suggested by FBA with i JN1463), acting as an ATP source. Alternatively, P. putida can produce NAD(P)H via the oxidation of glucose 6-phosphate, contributing to the intracellular pool of reducing equivalents [77]. The basis for these rearrangements was investigated by quantitative proteomics as disclosed in the next section.…”

Implementation of the β-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol

“…Finally, we observed increased levels of enzymes of the Entner-Doudoroff pathway (Edd, Eda) and several other glycolytic enzymes (ZwfA, Pgl, Gap, Pgm) in Δ gcl + BHAC. However, these changes are not easy to rationalize, as glycolytic pathways in P. putida KT2440 form a complex network (Nikel et al, 2015), and there are three different isoforms of Zwf with different cofactor specificities (Volke et al, 2021).…”

Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model

“…Insufficient activity of this multienzyme complex in P. putida (Ebert et al, 2011) might be exacerbated by other factors such as product inhibition with NADH (Bisswanger, 1974; Moxley and Eiteman, 2021). A massive flux of carbon through the direct phosphorylation route in GCDbglC_glf_lacY mutant may lead to the enhanced production of NADH in glucose 6-phosphate 1-dehydrogenase (Zwf) step ( Figure 1) (Volke et al, 2021) and lack of NAD - required for the pyruvate dehydrogenase reaction. Excess acetate (˃ 60 % of it, as shown by the model) likely comes from L-glutamate formed from α-ketoglutarate in the tricarboxylic acid cycle ( Figure 1 ) which is converted by the activities of NAD(P)-specific glutamate dehydrogenase (PP_2080, PP_0675), acetylornithine aminotransferase (PP_4481, PP_0372), and acetylornithine deacetylase (PP_5186, PP_3571).…”

“…Insufficient activity of this multienzyme complex in P. putida (Ebert et al, 2011) might be exacerbated by other factors such as product inhibition with NADH (Bisswanger, 1974;Moxley and Eiteman, 2021). A massive flux of carbon through the direct phosphorylation route in GCDbglC_glf_lacY mutant may lead to the enhanced production of NADH in glucose 6-phosphate 1-dehydrogenase (Zwf) step (Figure 1) (Volke et al, 2021) and lack of NADrequired for the pyruvate dehydrogenase…”