Abstract
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.
Highlights
Ornithine acetyltransferase (OATase,1 N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase; EC 2.3.1.35, encoded by the argJ gene in procaryotes) participates in arginine biosynthesis in microorganisms
Expressed Subunits of OATase Are Not Active—We previously showed that the two subunits of recombinant OATases from three thermophilic microorganisms, always co-purified by affinity chromatography irrespective of the N- or C-terminal His tag, respectively, fused to the ␣ or the  subunit [2]
Analysis of Thr-197-substituted Mutant Proteins in Vitro— Assuming that the invariant threonine (Thr-197 in B. stearothermophilus OATase) in the conserved ATML sequence could play an essential role in the intramolecular cleavage we replaced this amino acid by a serine, cysteine, or glycine residue
Summary
Strains and Materials—The argJ gene was cloned from the B. stearothermophilus NCIB 8224 strain [11]. We constructed an argJ variant with a translation stop codon for the ␣ subunit encoding sequence immediately followed by a ribosomebinding site preceding either 1 or 2 versions (see above). This preserved the single mRNA transcription but resulted in independent translation of the ␣ and  subunits (see Fig. 1B). Detection of Acetylated Proteins—Purified His-tagged OATases were incubated with 14C-labeled N-acetyl-L-glutamic acid as acetyl donor, but in the absence of acetyl acceptor (semi-reaction) in the above mentioned mixed buffer at 37 °C for 30 min or at 65 °C for 1 h, reaction products were separated on SDS-PAGE (a 12% gel). The samples were treated at 65 °C for 10 min and shortly centrifuged, and the supernatant was used for protein separation on SDS-PAGE
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