Effects of N-terminal deletions on 1-aminocyclopropane-1-carboxylate synthase activity.
A series of nested N-terminal deletions were made on the full-length (wt) and C-terminal deleted (Cdel) 1-aminocyclopropane-1-carboxylate synthase cDNAs. These wt and mutant ACC synthases were over-expressed in a heterologous E. coli expression system. It was found that removal of an amino acid region (residues 2-12) from the non-conserved N-termini of wt and Cdel ACC synthases led to a slight increase in both in vivo ACC production and in vitro ACC synthase activity. Further deletion of 11 amino acids through Glu-23 from the N-termini of both wt and Cdel ACC synthases resulted in a substantial reduction in both in vivo ACC production and in vitro enzyme activity. Deletion of an amino acid region, residues 3 through 27, from the N-terminus of ACC synthase abolished enzyme activity completely. Kinetic analysis of a highly purified double-deletion mutant (NCdel-1) of ACC synthase demonstrated that the Km of this mutant is 42 microM, which is much smaller than that of the corresponding Cdel (280 microM) and closer to that of wt (22 microM) reported previously, suggesting a clear effect of the non-conserved N-terminal region on its ACC synthase function.
Li
N
N
Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Huxtable
S
S
Yang
S F
SF
Kung
S D
SD
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase