Effects of N‐terminal deletions on 1‐aminocyclopropane‐1‐carboxylate synthase activity

FEBS Letters

Li, Ning¹  Huxtable, Susan¹ 

[1] Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

关键词: Tomato fruit;    ACC synthase;    Amino-terminus;    Deletion;    Kinetics;    aa;    amino acid;    ACC;    1-aminocyclopropane-1-carboxylic acid;    AdoMet;    S-adenosylmethionine;    DTT;    dithiothreitol;    SDSPAGE;    sodium dodecyl sulfate-polyacrylamide gel electrophoresis;    PCR;    polymerase chain reaction;    PMSF;    phenylmethanesulfonyl fluoride;    EDTA;    ethylenediaminetetraacetic acid;    EPPS;    N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid PLP;    pyridoxyl 5′phosphate;    IPTG;    isopropyl;    β-d-thiogalactopyranoside;    HEPES;    N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid;   

DOI  :  10.1016/0014-5793(95)01464-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 K _m of this mutant is 42 μM, which is much smaller than that of the corresponding Cdel (280 μM) and closer to that of wt (22 μM) reported previously, suggesting a clear effect of the non-conserved N-terminal region on its ACC synthase function.

【 授权许可】

Unknown