Open AccessIsolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase
Author(s) -
Raymond E. Sheehy,
Mamoru Honma,
Masatoshi Yamada,
Takuji Sasaki,
Belinda Martineau,
William R. Hiatt
Publication year - 1991
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.173.17.5260-5265.1991
Subject(s) - biology , peptide sequence , ecori , nucleic acid sequence , microbiology and biotechnology , escherichia coli , open reading frame , biochemistry , structural gene , 1 aminocyclopropane 1 carboxylic acid , gene , plasmid , biosynthesis
Pseudomonas sp. strain ACP is capable of growth on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed.