Properties, sequence, and synthesis in Escherichia coli of 1-aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus.
The plant hormone ethylene is generated from a unique precursor, 1-aminocyclopropane-1-carboxylate (ACC). In previous studies, ACC deaminase, which degrades ACC to alpha-ketobutyrate and ammonia, was found in four strains of Pseudomonas, characterized, and sequenced. To verify the wider distribution of ACC deaminase in microorganisms, we purified and sequenced ACC deaminase from the yeast Hansenula saturnus. The purified enzyme was active toward ACC, D-serine and dl-coronamic acid, indicating the same stereospecificity as the Pseudomonas enzyme, but unlike the bacterial enzyme it was not active toward beta-chloro-D-alanine and O-acetyl-D-serine. Analyses of peptides from proteolytic digests of the purified and modified ACC deaminase covered more than 90% of its amino acid sequence and showed a blocked N-terminal residue as N-acetylserine. A cDNA encoding the ACC deaminase was isolated from H. saturnus cells incubated in alpha-aminoisobutyrate medium, and sequenced. The yeast enzyme has 441 amino acid residues, of which 60 to 63% are identical to those of reported Pseudomonas enzymes. The open reading frame encoding ACC deaminase was subcloned into pET-11d and expressed in Escherichia coli BL21 (DE3) as an active enzyme.
Minami
R
R
Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, 060-8589, Japan.
Uchiyama
K
K
Murakami
T
T
Kawai
J
J
Mikami
K
K
Yamada
T
T
Yokoi
D
D
Ito
H
H
Matsui
H
H
Honma
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D019755:Carbon-Carbon Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D014162:Transfection
C033634:1-aminocyclopropane-1-carboxylate deaminase; D019755:Carbon-Carbon Lyases