Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase.
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.
Sheehy
R E
RE
Calgene, Inc. Davis, California 95616.
Honma
M
M
Yamada
M
M
Sasaki
T
T
Martineau
B
B
Hiatt
W R
WR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000598:Amino Acids, Cyclic; D001345:Autoradiography; D001483:Base Sequence; D015153:Blotting, Western; D019755:Carbon-Carbon Lyases; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006868:Hydrolysis; D008190:Lyases / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011549:Pseudomonas / Q000235:genetics*; D015183:Restriction Mapping
D000596:Amino Acids; D000598:Amino Acids, Cyclic; D004269:DNA, Bacterial; C023863:1-aminocyclopropane-1-carboxylic acid; C033634:1-aminocyclopropane-1-carboxylate deaminase; D008190:Lyases; D019755:Carbon-Carbon Lyases