Chain length determination of prenyltransferases: both heteromeric subunits of medium-chain (E)-prenyl diphosphate synthase are involved in the product chain length determination.
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.
Zhang
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Institute for Chemical Reaction Science, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.
Li
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Koyama
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D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D002384:Catalysis; D019281:Dimerization; D004122:Dimethylallyltranstransferase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D045782:Hemiterpenes; D007700:Kinetics; D016982:Micrococcus luteus / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000737:chemistry; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011106:Polyisoprenyl Phosphates / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012717:Sesquiterpenes
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C004809:isopentenyl pyrophosphate; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; D004122:Dimethylallyltranstransferase; C024844:trans-hexaprenyltranstransferase