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.
Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDH(I) was produced when cells were grown on isophthalate, while GDH(II) was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDH(P), when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDH(I) and GDH(II) were found to be homotetramers, while GDH(P) was found to be a homohexamer. GDH(II) was more sensitive to inhibition by isophthalate (2.5- and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDH(I). Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDH(I) and GDH(II), in strain PP4. In strain ISP4, irrespective of the carbon source, the GDH(P) produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDH(P) from isophthalate-grown cells was 2.5- to 3-fold higher than that of GDH(P) from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDH(P) in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.
Vamsee-Krishna
C
C
Biotechnology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai 400 076, India.
Phale
Prashant S
PS
D000150:Acinetobacter / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D018086:Glutamate Dehydrogenase (NADP+) / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010795:Phthalic Acids / Q000378:metabolism* / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*
D010795:Phthalic Acids; C059768:isophthalate; D018086:Glutamate Dehydrogenase (NADP+)