Bifunctional Enzymes

The argJ gene coding for N 2-acetyl-l-ornithine: l-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of l-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear 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-tagged ArgJ enzyme of M. jannaschii only catalyzes N 2-acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi±bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [ 14 C]acetylCoA or [ 14 C]N 2-acetyl-l-glutamate as acetyl donors. In this catalysis l-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of l-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate a and b subunits which appear to assemble to a2b2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences.

In Bacillus stearothermophilus ornithine acetyltransferase is a bifunctional enzyme, catalyzing the first and the fifth steps of arginine biosynthesis; it follows a pingpong kinetic mechanism. A single chain precursor protein is cleaved between the alanine and threonine residues in a highly conserved ATML sequence leading to the formation of ␣ and ␤ subunits that assemble into a heterotetrameric 2␣2␤ molecule. The ␤ subunit has been shown to form an acetylated intermediate in the course of the transacetylation reaction. The present data show that the precursor protein synthesized in vitro or in vivo undergoes a self-catalyzed cleavage involving an invariant threonine (Thr-197). Using site-directed mutagenesis T197G, T197S, and T197C derivatives have been generated. The T197G substitution abolishes both precursor protein cleavage and catalytic activity, whereas T197S and T197C substitutions reduce precursor cleavage and catalytic activity in the order Thr-197 (wild type) 3 Ser-197 3 Cys-197. A mechanism is proposed in which Thr-197 plays a crucial role in the autoproteolytic cleavage of ornithine acetyltransferase.

The argJ gene coding for N 2 -acetyl-l-ornithine: l-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of l-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear 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-tagged ArgJ enzyme of M. jannaschii only catalyzes N 2 -acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi±bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [ 14 C]acetylCoA or [ 14 C]N 2 -acetyl-l-glutamate as acetyl donors. In this catalysis l-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of l-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate a and b subunits which appear to assemble to a2b2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences.

In Bacillus stearothermophilus ornithine acetyltransferase is a bifunctional enzyme, catalyzing the first and the fifth steps of arginine biosynthesis; it follows a pingpong kinetic mechanism. A single chain precursor protein is cleaved between the alanine and threonine residues in a highly conserved ATML sequence leading to the formation of ␣ and ␤ subunits that assemble into a heterotetrameric 2␣2␤ molecule. The ␤ subunit has been shown to form an acetylated intermediate in the course of the transacetylation reaction. The present data show that the precursor protein synthesized in vitro or in vivo undergoes a self-catalyzed cleavage involving an invariant threonine (Thr-197). Using site-directed mutagenesis T197G, T197S, and T197C derivatives have been generated. The T197G substitution abolishes both precursor protein cleavage and catalytic activity, whereas T197S and T197C substitutions reduce precursor cleavage and catalytic activity in the order Thr-197 (wild type) 3 Ser-197 3 Cys-197. A mechanism is proposed in which Thr-197 plays a crucial role in the autoproteolytic cleavage of ornithine acetyltransferase.

In Bacillus stearothermophilus ornithine acetyltransferase is a bifunctional enzyme, catalyzing the first and the fifth steps of arginine biosynthesis; it follows a ping-pong kinetic mechanism. A single chain precursor protein is cleaved between the alanine and threonine residues in a highly conserved ATML sequence leading to the formation of alpha and beta subunits that assemble into a heterotetrameric 2alpha2beta molecule. The beta subunit has been shown to form an acetylated intermediate in the course of the transacetylation reaction. The present data show that the precursor protein synthesized in vitro or in vivo undergoes a self-catalyzed cleavage involving an invariant threonine (Thr-197). Using site-directed mutagenesis T197G, T197S, and T197C derivatives have been generated. The T197G substitution abolishes both precursor protein cleavage and catalytic activity, whereas T197S and T197C substitutions reduce precursor cleavage and catalytic activity in the order Thr-197 (wild type) --> Ser-197 --> Cys-197. A mechanism is proposed in which Thr-197 plays a crucial role in the autoproteolytic cleavage of ornithine acetyltransferase.

The argJ gene coding for N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of L-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear 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-tagged ArgJ enzyme of M. jannaschii only catalyzes N2-acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [14C]acetylCoA or [14C]N2-acetyl-L-glutamate as acetyl donors. In this catalysis L-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of L-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate alpha and beta subunits which appear to assemble to alpha2beta2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences.