Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus.

A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes.

Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase.

A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcloned B. stearothermophilus argJ can complement a double argA argE E. coli mutant to prototrophy. Gel-filtration of a heat-treated extract of the complemented double mutant E. coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M(r) 110,000. Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine. These results suggest that both activities can be ascribed to a single protein.