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.
Sakanyan
V
V
Pharmagen, Yerevan, Republic of Armenia.
Desmarez
L
L
Legrain
C
C
Charlier
D
D
Mett
I
I
Kochikyan
A
A
Savchenko
A
A
Boyen
A
A
Falmagne
P
P
Pierard
A
A
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D018076:DNA, Complementary; D000581:Amidohydrolases; C019672:aminoacylase I