GjUGT2 amino acid sequence as a query. From an initial list of UGT candidates obtained by BLASTX search, the contigs belonging for the group G of PSPGs (Nagatoshi et al., 2011) have been manually selected to provide the master candidate list, as shown in Supplemental Table 3 on the net. We developed genespecific primers and isolated a fulllength cDNA corresponding for the contig Cr8440 as UGT8. The nucleotide sequences of PCR primers made use of for RACE PCR and amplification of fulllength cDNAs of UGTs are shown in Supplemental Tables four and five on the net, respectively. Heterologous Expression of UGTs The UGT6, 7, and 8 cDNA fragments containing their respective open reading frames had been amplified by PCR with Thermococcus kodakaraensis FX DNA polymerase (Toyobo) making use of the PCR primers containing acceptable restriction web sites (see Supplemental Table six on-line). The PCR goods had been cloned in to the pMD20 Tvector to confirm their sequences then subcloned into pQE30 vector (Qiagen) to create Nterminal fusion proteins with a His6 tag.2611225-93-3 web Escherichia coli strain JM109 was utilized as the host for expression. Transformed cells were cultured at 37 until the absorbance at 600 nm reached 0.six and then additional incubated overnight at 30 before harvest. The recombinant proteins have been affinity purified on a nickelnitrilotriacetic acid agarose matrix (Qiagen) based on the manufacturer’s guidelines. Protein content material within the enzyme preparations was estimated working with the approach of Bradford (1976).Enzyme AssaysChemicals 7Deoxyloganin tetraacetate was kindly provided by K. Inoue (Yokohama College of Pharmacy). 7Deoxyloganin and 7deoxyloganic acid were ready from 7deoxyloganin tetraacetate according to the technique described previously (Nagatoshi et al., 2011). Loganetin, 7deoxyloganetin, and 7deoxyloganetic acid have been enzymatically ready from loganin, 7deoxyloganin, and 7deoxyloganic acid, respectively, as follows. A 20mg aliquot of every single glucoside was dissolved in ten mL of 50 mM phosphatecitrate buffer, pH four.eight, containing 50 units/mL almond bglucosidase (SigmaAldrich) and incubated for 3 h at 37 . Immediately after centrifugation, the reaction mixture was extracted with ethyl acetate three times. The combined ethyl acetate extract was concentrated below lowered stress to yield the aglycone. The purity of every single aglycone therefore obtained was estimated by quantitative 1H NMR analysis (Hasada et al., 2011). Geniposide, genipin, and loganin had been obtained from Wako Pure Chemical substances and secologanin from SigmaAldrich. Iridotrial was synthesized by N. Kato (Nagoya City University). All other chemical compounds had been of industrial reagentgrade excellent unless otherwise stated.Price of 5458-56-0 The normal reaction mixture for the enzyme assays (total volume of 50 mL) contained 50 mM TrisHCl, pH 8.PMID:23800738 0, 5 mM UDPGlc, 1 mM acceptor substrates, as well as the enzyme preparation. The reaction was performed at 30 then terminated by the addition of 100 mL of methanol. Soon after centrifugation at 12,000g for five min, the reaction items were analyzedThe Plant Cellby reversedphase HPLC, and elution was monitored employing a photodiode array detector. To separate the substrates and their glucosides, the following gradient elution programs had been utilized: for iridoid glucosides, 0 to 16 min, 30 to 60 methanol and 16 to 19 min, 60 methanol; for flavonoids, coumarins, and various phenolic glucosides, 0 to 26 min, 15 to 52 acetonitrile, 26 to 29 min, 52 to one hundred acetonitrile, and 29 to 33 min, one hundred acetonitrile. To identify the kinetic parameters, enzyme assay.
