| Research Abstract |
Prenyltrransferases accepting aromatic substrates occur in a wide range from bacteria to human, which play important roles in biosyntheses of quinone compounds mediating electron transfer, and also in those of many plant secondary metabolites. However, the discovery of genes encoding these enzymes and molecular analyses of them were hardly reported., because most of them are membrane-bound proteins. In this study, we used p-hydroxybenzoic acid : prenyltransferase as a model, e.g. LePGT involved in naphthoquinone biosynthesis and Coq2 for ubiquinone biosynthesis, and made attempts to elucidate their biochemical and enzymatic reaction mechanism.
First we made an attempt to make a chimeric protein between soluble farnesyl diphosphate synthase whose crystal structure was elucidated and the membrane-bound LePGT, to make a soluble prenyltransferase, which accepts p-hydroxybenzoate as the substrate. This trial was, however, unsuccessful because the chimeric proteins did not show clear prenyltn
… More
rasferase activity, although some of them were highly expressed in E.coli.
After many divergent approaches, we finally succeeded to express LePGT of Lithospermum erythrorhizon in the Baculovirus system in an appreciable amount. This protein, which has 9 transmembrane α-helices, was not influenced in the enzymatic activity if His-tag was attached at the C-termimus, and the fusion protein expressed in Sf9 cells were recovered from the microsomal fraction, which was then efficiently solubilized by sodium deoxycholate. This solubilized recombinant protein was affinity-purified with Ni-agarose column to be a single band in the SDS PAGE. We have been improving this protocol for the supply to crystallization experiment of the membrane protein.
We have been trying to determine critical amino acids for the enzymatic function by introducing point mutation into LePGT protein. This site-directed mutagenesis study showed that the conserved sequences containing either NDxxD motif or YAHQD motif among several hydrophilic loops are important for the enzymatic activity, and some critical amino acids for the enzymatic function have been determined. It is presumed that the former motif is responsible for prenyl substrate recognition and the latter is for aromatic substrate. Now, the Km values of mutant enzymes are analyzed in order to identify the substrate binding sites.
In the process of analyzing the LePGT ortholog, yeast Coq2, it was fond that the high expression of this prenyltransferase increased the accumulation of ubiquinone (Coenzyme Q) to tow fold both in yeast and in plant. Furthermore if the subcellular localization is altered from mitochondria, the native location, to endoplasmic reticulum, higher effect on the increase of ubiquinone production was observed. Less
|
