Characterization and kinetic mechanism of mono- and bifunctional ornithineacetyltransferases from thermophilic microorganisms

The argJ gene coding for N-2-acetyl-L-ornithine: L-glutamate N-acetyltransf erase, the key enzyme involved in the acetyl cycle of L-arginine biosynthes is, has been cloned from thermophilic procaryotes: the archaeon Methanoccoc us jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearot hermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the lin ear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His-tagge d ArgJ enzyme of M. jannaschii only catalyzes N-2-acetylornithine conversio n to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA a s the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme , whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunct ional. Kinetic data demonstrate that in all three thermophilic organisms Ar gJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [C-14]acetylCoA or [C-14]N-2-acetyl-L-glutamate as acetyl donors. In this catalysis L-ornithi ne acts as an inhibitor; this amino acid therefore appears to be a key regu latory molecule in the acetyl cycle of L-arginine synthesis. Thermophilic A rgJ are synthesized as protein precursors undergoing internal cleavage to g enerate alpha and beta subunits which appear to assemble to alpha 2 beta 2 heterotetramers in E. coli. The cleavage occurs between alanine and threoni ne residues within the highly conserved PXM-ATML motif detected in all avai lable ArgJ sequences.