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

Autor: Chen X; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu 241000, China., Wei W; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu 241000, China., Xiong W; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu 241000, China., Wu S; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu 241000, China., Wu Q; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu 241000, China., Wang P; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu 241000, China., Zhu G; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, Anhui Normal University, Wuhu 241000, China.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2023 Oct 07; Vol. 24 (19). Date of Electronic Publication: 2023 Oct 07.
DOI: 10.3390/ijms241914985
Abstrakt: 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 .
Competing Interests: The authors have no conflict of interest to declare that are relevant to the content of this article.
Databáze: MEDLINE
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