The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans: molecular cloning, nucleotide sequence, and expression in Escherichia coli.
A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli.
Chou
J C
JC
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park 20742, USA.
Mulbry
W W
WW
Cohen
J D
JD
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004754:Enterobacter / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000581:Amidohydrolases; C114673:indole-3-acetyl-L-aspartic acid hydrolase, Pantoea agglomerans