Structural and functional relationship of ACC synthase

Ethylene is a plant hormone regulating many aspects of plant growth and development, and its effects are of commercial importance in both agriculture and horticulture. It has been established that ethylene produced in higher plants is synthesized from S-adenosylmethionine (AdoMet) via l-aminocyclopropane-l-carboxylic acid (ACC), and the reactions are catalyzed by ACC synthase and ACC oxidase. The rate-limiting step is at the formation of ACC controlled by ACC synthase. The lability of ACC synthase and its low abundance in plant tissues led to difficulties in purification and limited characterization of the enzyme. Molecular cloning and expression of genes encoding ACC synthase in heterologous systems provided an opportunity to explore purification and analysis of structure-function aspects of this enzyme. ACC synthase is a member of PLP-dependent enzymes. It shares with other PLP-dependent enzymes a number of highly conserved amino acid residues which are believed to be responsible for PLP-binding. Two of these amino acids, K278 and R286, were changed by site-directed mutagenesis and mutants were analyzed by biophysical and biochemical methods. Mutants were made from mutagenic primers and expressed in an E. coli expression system. High levels of soluble expression were achieved, and proteins were purified to homogeneity through a two-step purification system. Kinetic analysis of these mutants showed that they were of lower affinity toward the coenzyme PLP and lower catalytic efficiency than the control. ACC synthase exists either as a dimer or a monomer in plant cell extract. The recombinant ACC synthase of zucchini purified from E. coli soluble extract was shown to be a dimer. ACC synthase of zucchini refolded from insoluble inclusion bodies from E.coZi was also shown to be a dimer with a similar native molecular weight and kinetic parameters to that of the soluble form