Carbon source-dependent modulation of NADP-glutamate dehydrogenases in isophthalate-degrading Pseudomonas aeruginosa strain PP4, Pseudomonas strain PPD and Acinetobacter lwoffii strain ISP4

Abstract

Acinetobacter lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeruginosa strain PP4 and Pseudomonas strain PPD. Isophthalate has been reported to be a potent competitive inhibitor of glutamate dehydrogenase (GDH). Exogenous supplementation of isophthalate with glutamate or a-ketoglutarate at 1 mM concentration caused strains PP4 and PPD to grow faster than in the presence of isophthalate alone; however, no such effect was observed in strain ISP4. When grown on isophthalate, all strains showed activity of NADPdependent GDH (NADP-GDH), while cells grown on glucose, 2¾ yeast extract-tryptone broth (2YT) or glutamate showed activities of both NAD-dependent GDH (NAD-GDH) and NADP-GDH. Activity staining, inhibition and thermal stability studies indicated the carbon sourcedependent presence of two (GDH I and GDH II ), three (GDH A , GDH B and GDH C ) and one (GDH P ) forms of NADP-GDH in strains PP4, PPD and ISP4, respectively. The results demonstrate the carbon source-dependent modulation of different forms of NADP-GDH in these bacterial strains. This modulation may help the efficient utilization of isophthalate as a carbon source by overcoming the inhibitory effect on GDH.

Bypassing Isophthalate Inhibition by Modulating Glutamate Dehydrogenase (GDH): Purification and Kinetic Characterization of NADP-GDHs from Isophthalate-Degrading Pseudomonas aeruginosa Strain PP4 and Acinetobacter lwoffii Strain ISP4

“…NADP-dependent GDH I and GDH II were purified to homogeneity from P. aeruginosa strain PP4 grown on isophthalate and glucose, respectively, while GDH P was purified from A. lwoffii strain ISP4 grown on either isophthalate or 2YT. Incubation at 60°C was included as a step during purification of GDH I and GDH P (33). The enzyme yields were better in the presence of ␣-KG (1 mM), EDTA (1 mM), and glycerol (4.35%).…”

“…GDH assay and purification. The enzyme activity was monitored spectrophotometrically (Perkin-Elmer, United States) by measuring the decrease in the absorbance at 340 nm as described previously (33). The reaction mixture (1 ml) contained ␣-KG (5 mM), ammonium chloride (200 mM), NADPH (0.1 mM), and an appropriate amount of enzyme in HEPES-NaOH buffer (100 mM, pH 8.5).…”

“…Since it is known that isophthalate is a competitive inhibitor of GDH, the question was how these bacterial strains are able to grow on isophthalate, avoid inhibition of GDH by isophthalate, and synthesize glutamate family amino acids. Carbon source-dependent NADP-GDH activities, sensitivity to inhibition by isophthalate, activity staining, and thermal stability studies suggested that (i) strain PP4 produced GDH I when it was grown on isophthalate and GDH II when it was grown on glucose and (ii) irrespective of the carbon source strain ISP4 produced only one form of GDH, GDH P (33). Since this was the first study of carbon source-dependent modulation of NADP-GDH in a bacterial system, the goal was to purify GDH I , GDH II , and GDH P and determine the biochemical and kinetic properties of these enzymes.…”

“…Compared to the organisms whose metabolic pathways for isophthalate degradation have been studied, a large number of organisms have been studied in detail to determine their metabolic pathways for phthalate and terephthalate degradation (12,17,18,20,25,26,29,31,32,35,36). The fewer isophthalate-degrading strains and the difficulties in isolating them could be due to the fact that isophthalate acts as a competitive inhibitor of glutamate dehydrogenase (GDH), which plays an important role at the interface of C metabolism and N metabolism (5,11,13,16,27,28,30,33,34). GDH performs oxidative deamination of glutamate to ␣-ketoglutarate (␣-KG) and reductive amination of ␣-KG to glutamate, and depending on the cofactor requirement the enzyme is either NAD-, NADP-, or NAD(P)-GDH (7,19).…”

“…Activity staining was performed by resolving appropriate amounts of purified proteins on a native PAGE (7.5%) gel at a constant current of 4 mA at 4°C, which was followed by activity staining as described previously (33).…”