Molecular analysis and functional change of cis-isoprene-chain elongation enzyme

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(1)In complimentary experiments using a yeast mutant #64 with low conversion of polyprenol to dolichol, the following three clones, #64-1、 #64-2、#64-4 were screened. In these yeasts shorter C50-60 polyprenols were detected together with longer dolichols.(2)Three biotinylated prenals (farnesal, C55-polyprenal, and C80-polyprenal) were chemically synthesized and their effects on in vitro polyprenol reduction reaction were analyzed. The C-80compound alone showed inhibitory effect and the effect was specific compared with those of Triton X-100, Chaps, octylglucoside, deoxycholic acid, and Tween 80. By SDS elctrophoresis, a protein (50,000, Da) associated with the C80-compound was detected.(3)Two ferns (warabi and zenmai in Japanese) were analyzed to both contain C80, C85, C90 dolichols. A seaweed (Wakame in Japanese) was also analyzed to contain C90,C95,C100 polyprenols and C80, C85, C90 dolichols.(4)All-trans-geranylgeraniol (GGOH) was converted to 2,3-dihydrogeranylgeranoic acid (2,3-diGGA) using rat thymic cells and human HL-60 cells. The 2,3-diGGA showed the ability to induce apoptosis of HL-60 cells in the presence of 0.1% FBS. In the copresence of a caspase-3 inhibior, the apoptosis was repressed and lipid droplets were observed. On the other hand, the same compound showed the ability to induce lipid droplets in the presence of 10% FBS. In the copresence of an acyl-CoA synthethase inhibitor, the lipid droplets formation was repressed and apoptosis was observed. GGA also inducded the apoptosis in 0.1% FBS, but the HL-60 cells were differentiated to neutrophiles in 10% FBS.(5)The study using mutated bacterial undecaprenyl diphosphatesynthase, showd the importance of Arg residue in the structure of P-loop hand Asp29, Arg33, Arg80 residies for catalytic activiy. Also, Tyr71,Tyr148, Ty210 residues were important for the binding of isopentenyl diphosphate and farnesyl diphosphat.