Arginine Biosynthesis inThermotoga maritima: Characterization of the Arginine-SensitiveN-Acetyl-l-Glutamate Kinase

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Abstract
To help clarify the control of arginine synthesis inThermotoga maritima, the putative gene (argB) forN-acetyl-l-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is inT. maritimathe feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of ∼4, and the 50% inhibitory arginine concentration (I0.5) was shown to increase with temperature, approaching above 65°C the I0.50observed at 37°C with the mesophilic NAGK ofPseudomonas aeruginosa(the best-studied arginine-inhibitable NAGK). At 75°C, the inhibition by arginine ofT. maritimaNAGK was due to a large increase in theKmfor acetylglutamate triggered by the inhibitor, but at 37°C arginine also substantially decreased theVmaxof the enzyme. The NAGKs ofT. maritimaandP. aeruginosabehaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast,Escherichia coliNAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants ofT. maritimaNAGK are also discussed.

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