Heterologous expression and site-directed mutagenesis of the 1-aminocyclopropane-1-carboxylate oxidase from kiwi fruit.
A system has been developed for the expression in Escherichia coli of 1-aminocyclopropane-1-carboxylate (ACC) oxidase from kiwi fruit. In this first report of site-directed mutagenesis of ACC oxidase, seven different mutants of the enzyme have been expressed, and their activities compared to that of the heterologoulsy expressed wild-type enzyme. No great loss of activity was observed when Lys172 was substituted by either Ala or Cys, or when Gly137 was substituted by Pro. However, the mutant proteins showed only 1% of the wild-type activity when substitutions were made of Asp179, His177, and Lys158. The results are discussed in terms of possible mechanisms by which ACC oxidase is activated by carbon dioxide, and in terms of structural motifs suggested by the known structure of isopenicillin N-synthase, an enzyme related by mechanism and sequence similarity to ACC oxidase. It is concluded that Lys172, a putative carbon dioxide binding site, has no role to play in the catalytic activity of the enzyme. The results support a previous suggestion that ACC oxidase shares important structural features with isopenicillin N-synthase.
Lay
V J
VJ
Zeneca Agrochemicals, Jealott's Hill Research Station, Bracknell, UK.
Prescott
A G
AG
Thomas
P G
PG
John
P
P
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005638:Fruit / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase