It has been proposed that NADP+-specificity of isocitrate dehydrogenase (ICDH) evolved as an adaptation of microorganisms to grow on acetate as the sole source of carbon and energy. In Escherichia coli, changing the cofactor specificity of ICDH from NADP+ to NAD+ (cofactor swapping) decreases the growth rate on acetate. However, the metabolic basis of this phenotype has not been analyzed. In this work, we used constraint-based modeling to investigate the effect of the cofactor swapping of ICDH in terms of energy production, response of alternative sources of NADPH, and partitioning of fluxes between ICDH and isocitrate lyase (ICL) -a crucial bifurcation when the bacterium grows on acetate-. We generated E. coli strains expressing NAD+-specific ICDH instead of the native enzyme, and bearing the deletion of the NADPH-producing transhydrogenase PntAB. We measured their growth rate and acetate uptake rate, modeled the distribution of metabolic fluxes by Flux Balance Analysis (FBA), and quantified the specific activities of NADPH-producing dehydrogenases in central pathways. The cofactor swapping of ICDH led to one-third decrease in biomass yield, irrespective of the presence of PntAB. According to our simulations, the diminution in growth rate observed upon cofactor swapping could be explained by one-half decrease in the total production of NADPH and a lower availability of carbon for biosynthesis because of a change in the partition at the isocitrate bifurcation. Together with an increased total ATP production, this scenario resulted in a 10-fold increment in the flux of ATP not used for growing purposes. PntAB was identified as the primary NADPH balancing response, with the dehydrogenases of the oxidative branch of the Pentose Phosphate Pathway and the malic enzyme playing a role in its absence. We propose that in the context of E. coli growing on acetate, the NADP+-specificity of ICDH is a trait that impacts not only NADPH production, but also the efficient allocation of carbon and energy.

Chile has the highest per capita intake of sugar-sweetened beverages (SSB) world-wide. However, it is unknown whether the effects from this highly industrialized food will mimic those reported in industrialized countries or whether they will be modified by local lifestyle or population genetics. Our goal was to evaluate the association between SSB intake and fasting glucose in the Chilean population. We calculated a weighted genetic risk score (GRSw) based on 16 T2D risk SNPs in 2828 non-diabetic participants of the MAUCO cohort. SSB intake was categorized in four levels using a food frequency questionnaire. Log-fasting glucose was regressed on SSB and GRSw tertiles while accounting for socio-demography, lifestyle, obesity, and Amerindian ancestry. Fasting glucose increased systematically per unit of GRSw (β = 0.02 ± 0.006, p = 0.00002) and by SSB intake (β[cat4] = 0.04 ± 0.009, p = 0.0001), showing a significant interaction, where the strongest effect was observed in the highest GRSw-tertile and in the highest SSB consumption category (β = 0.05 ± 0.02, p = 0.02). SNP-wise, SSB interacted with additive effects of rs7903146 (TCF7L2) (β = 0.05 ± 0.01, p = 0.002) and with the G/G genotype of rs10830963 (MTNRB1B) (β = 0.19 ± 0.05, p = 0.001). Conclusions: The association between SSB intake and fasting glucose in the Chilean population without diabetes is modified by T2D genetic susceptibility.

Chile has high incidence rates of gallbladder cancer globally, particularly among Amerindian women, who also have a high prevalence of gallstones. We examined differences in inflammatory biomarkers between Mapuche and non-Mapuche women from the Chile Biliary Longitudinal Study, a cohort of women with ultrasound-detected gallstones. We randomly selected 200 Mapuche women frequency matched to non-Mapuche women on age and statin use Inflammatory biomarkers were analyzed using a multiplex assay and linear regression to assess associations of a priori markers (CCL20, CXCL10, IL-6, and IL-8) with ethnicity. Novel biomarkers were analyzed using exploratory factor analysis (EFA) and sufficient dimension reduction (SDR) to identify correlated marker groups, followed by linear regression to examine their association with ethnicity. The mean values of IL-8 were higher in Mapuche than non-Mapuche women (P = 0.04), while CCL20, CXCL10, and IL-6 did not differ significantly by ethnicity. EFA revealed two marker groups associated with ethnicity (P = 0.03 and P < 0.001). SDR analysis confirmed correlation between the biomarkers and ethnicity. We found higher IL-8 levels among Mapuche than non-Mapuche women. Novel inflammatory biomarkers were correlated with ethnicity and should be studied further for their role in gallbladder disease. These findings may elucidate underlying ethnic disparities in gallstones and carcinogenesis among Amerindians.

Background: The enzyme 6-phosphogluconate dehydrogenase (6PGDH) is the central enzyme of the oxidative pentose phosphate pathway. Members of the 6PGDH family belong to different classes: either homodimeric enzymes assembled from long-chain subunits or homotetrameric ones assembled from short-chain subunits. Dimeric 6PGDHs bear an internal duplication absent in tetrameric 6PGDHs and distant homologues of the β-hydroxyacid dehydrogenase (βHADH) superfamily. Methods: We use X-ray crystallography to determine the structure of the apo form of the 6PGDH from Gluconobacter oxydans (Go6PGDH). We carried out a structural and phylogenetic analysis of short and long-chain 6PGDHs. We put forward an evolutionary hypothesis explaining the differences seen in oligomeric state vs. dinucleotide preference of the 6PGDH family. We determined the cofactor preference of Go6PGDH at different 6-phosphogluconate concentrations, characterizing the wild-type enzyme and three-point mutants of residues in the cofactor binding site of Go6PGDH. Results: The structural comparison suggests that the 6PG binding site initially evolved by exchanging C-terminal α-helices between subunits. An internal duplication event changed the quaternary structure of the enzyme from a tetrameric to a dimeric arrangement. The phylogenetic analysis suggests that 6PGDHs have spread from Bacteria to Archaea and Eukarya on multiple occasions by lateral gene transfer. Sequence motifs consistent with NAD+- and NADP+-specificity are found in the β2-α2 loop of dimeric and tetrameric 6PGDHs. Site-directed mutagenesis of Go6PGDH inspired by this analysis fully reverses dinucleotide preference. One of the mutants we engineered has the highest efficiency and specificity for NAD+ so far described for a 6PGDH. Conclusions: The family 6PGDH comprises dimeric and tetrameric members whose active sites are conformed by a C-terminal α-helix contributed from adjacent subunits. Dimeric 6PGDHs have evolved from the duplication-fusion of the tetrameric C-terminal domain before independent transitions of cofactor specificity. Changes in the conserved β2-α2 loop are crucial to modulate the cofactor specificity in Go6PGDH.