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Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications.

ABSTRACT: Pseudomonas aeruginosa PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP⁺-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in Escherichia coli and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP⁺ and isocitrate, with lower Km values when compared to PaIDH2. Moreover, PaIDH1 possessed higher temperature tolerance (50 °C) and wider pH range tolerance (7.2-8.5) and could be phosphorylated. After treatment with the bifunctional PaIDH kinase/phosphatase (PaIDH K/P), PaIDH1 lost 80% of its enzymatic activity in one hour due to the phosphorylation of Ser115. Small-molecule compounds like glyoxylic acid and oxaloacetate can effectively inhibit the activity of PaIDHs. The mutant PaIDH1-D346I347A353K393 exhibited enhanced affinity for NAD⁺ while it lost activity towards NADP⁺, and the Km value (7770.67 μM) of the mutant PaIDH2-L589 I600 for NADP⁺ was higher than that observed for NAD⁺ (5824.33 μM), indicating a shift in coenzyme specificity from NADP⁺ to NAD⁺ for both PaIDHs. The experiments demonstrated that the mutation did not alter the oligomeric state of either protein. This study provides a foundation for the elucidation of the evolution and function of two NADP-IDHs in the pathogenic bacterium P. aeruginosa.

SUBMITTER: Chen X

PROVIDER: S-EPMC10574006 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications.

Chen Xuefei X Wei Wei W Xiong Wei W Wu Shen S Wu Quanchao Q Wang Peng P Zhu Guoping G

International journal of molecular sciences 20231007 19

Pseudomonas aeruginosa PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP+-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in Escherichia coli and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP+ and isocitrate, with lower Km values when compared to PaIDH2. Moreover, Pa ...[more]

PMID: 37834433

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Project description:Lay summaryWe probed the evolutionary robustness of two antivirulence drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine in the opportunistic pathogen Pseudomonas aeruginosa. Using an experimental evolution approach in human serum, we showed that antivirulence treatments are not evolutionarily robust per se, but vary in their propensity to select for resistance.Background and objectivesTreatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that antivirulence treatments disarm rather than kill pathogens, is controversial. Here, we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa.MethodologyWe subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance.ResultsWe found that mutants resistant against antivirulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in upp, a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance was presumably hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike.Conclusions and implicationsOur work highlights that antivirulence treatments are not evolutionarily robust per se. Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.

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Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications.

Publications

Two Different Isocitrate Dehydrogenases from <i>Pseudomonas aeruginosa</i>: Enzymology and Coenzyme-Evolutionary Implications.

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