Arginine Biosynthesis in Thermotoga maritima : Characterization of the Arginine-Sensitive N -Acetyl- l -Glutamate Kinase

Abstract

To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-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 in T. maritima the 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 ϳ4, and the 50% inhibitory arginine concentration (I 0.5 ) was shown to increase with temperature, approaching above 65°C the I 0.50 observed at 37°C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75°C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the K m for acetylglutamate triggered by the inhibitor, but at 37°C arginine also substantially decreased the V max of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved 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 coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.Thermotoga maritima, one of the most highly thermophilic eubacteria (optimum growth temperature, 80°C) (20) and possibly one of the deepest branching and more slowly evolving of the eubacterial lineages (2), has been the subject of an already completed genome sequencing project (33) and, given its possible evolutionary position and biotechnological potential (19), is also the target of one of the few structural genomics projects being developed now (25).To exploit to its maximum the completeness of the genomic information and the massive structural data expected, it would be highly desirable to have a detailed knowledge of the physiology and metabolism of T. maritima rather than the patchy existing knowledge (3). Concerning the object of the present work, arginine biosynthesis, crude T. maritima extracts were shown to exhibit (36) enzyme activity

Complex Formation and Catalytic Activation by the PII Signaling Protein of N-Acetyl-l-glutamate Kinase from Synechococcus elongatus Strain PCC 7942

“…The sigmoidal inhibition curve of NAGK activity by arginine that we observed for noncomplexed NAGK has a similar Hill coefficient to that of T. maritima, indicating a similar number of allosteric arginine inhibition sites on both NAGKs. Considering the hexameric structure of both enzymes, this supports the suggestion by Fernandez-Murga et al (37) that "the hexameric architecture may be a key determinant of arginine sensitivity among NAGKs." Complex formation with P II has a strong impact on NAGK arginine feedback inhibition, with the I 0.5 increasing by an order of magnitude and the Hill coefficient decreasing to about 3.…”

“…Arginine inhibition of NAGK from T. maritima was shown to be sigmoidal with a Hill coefficient of ϳ4, which indicates multiple cooperative allosteric inhibition sites for arginine (37). The sigmoidal inhibition curve of NAGK activity by arginine that we observed for noncomplexed NAGK has a similar Hill coefficient to that of T. maritima, indicating a similar number of allosteric arginine inhibition sites on both NAGKs.…”

Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms

“…Since the completion of a number of genome sequencing projects, a few instances indeed have emerged where no NAGS, but a bifunctional OAT, appears to be present. This is the case in B. subtilis (reinterpretation of old complementation data [55,81]), G. stearothermophilus, and Thermotoga spp., assuming that T. maritima, whose genome was completely sequenced, is similar to T. neapolitana, which has a bifunctional OAT (26,43). The lack of biochemical information on the catalytic properties of most genomically identified OATs precludes further identification.…”

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…Six other genes among the cohort were highly expressed and have putative functional links to NR, NO 3 2 assimilation, or the integration of N-and C-metabolism. They include (1) a second putative NADH-NO 2 2 reductase (NiRBD, EG02286) (Bowler et al, 2008) targeted to the chloroplast, which is similar to NirB found in fungi and bacteria; (2) a voltage-gated, vacuolar NO 3 -(chloride channel [ClC] family) transporter (Phatr3_EG01952); (3) an acetylornithine transaminase (ACOAT, J50577), a chloroplasttargeted protein producing N-acetylornithine and 2-oxoglutarate (2-OG) from glutamic precursors (Fernandez-Murga et al, 2004); (4) isocitrate dehydrogenase (ICDH; J24195), the third enzyme in the TCA cycle and primary producer of 2-OG; (5) carbamoyl phosphate synthetase (unCPS, J24195), a key component of the ornithine-urea pathway (Allen et al, 2011); and (6) a molybdenum cofactor biosynthesis protein (MoaC, J15625), a subunit of molybdopterin synthase, in which molybdopterin is the required cofactor necessary for NO 3 2 binding and electron transfer in the NR active site (Figures 7C and 7D; Supplemental Table 1A). Of the other upregulated NR cohort genes, 10 have lower reads per kilobase million (RPKM) values and 13 are not associated with statistically significant annotations beyond "hypothetical protein.…”