| pubmed-article:11027152 | pubmed:abstractText | Among prenyltransferases, medium-chain (E)-prenyl diphosphate synthases are unusual because of their heterodimeric structures. The larger subunit has highly conserved regions typical of (E)-prenyltransferases. The smaller one has recently been shown to be involved in the binding of allylic substrate as well as determining the chain length of the reaction product [Zhang, Y.-W., et al. (1999) Biochemistry 38, 14638-14643]. To better understand the product chain length determination mechanism of these enzymes, several amino acid residues in the larger subunits of Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase and Bacillus subtilis heptaprenyl diphosphate synthase were selected for substitutions by site-directed mutagenesis and examined by combination with the corresponding wild-type or mutated smaller subunits. Replacement of the Ala at the fifth position upstream to the first Asp-rich motif with bulky amino acids in both larger subunits resulted in shortening the chain lengths of the major products, and a double combination of mutant subunits of the heptaprenyl diphosphate synthase, I-D97A/II-A79F, yielded exclusively geranylgeranyl diphosphate. However, the combination of a mutant subunit and the wild-type, I-Y103S/II-WT or I-WT/II-I76G, produced a C(40) prenyl diphosphate, and the double combination of the mutants, I-Y103S/II-I76G, gave a reaction product with longer prenyl chain up to C(50). These results suggest that medium-chain (E)-prenyl diphosphate synthases take a novel mode for the product chain length determination, in which both subunits cooperatively participate in maintaining and determining the product specificity of each enzyme. | lld:pubmed |
