Cloning And Characterization Of Phosphomevalonate Kinase Gene From Poria Cocos

Poria cocos(Schw.) Wolf is a higher fungus, its sclerotium is edible and an important traditional Chinese medicine. The epidermis and the inner part of the dried sclerotium of P. cocos have been used as two traditional Chinese medicines called Fu-Ling and Fu-Ling-Pi in Chinese, respectively. Triterpenoids are major bioactive constituents in P. cocos, they exhibit anti-tumor, anti-oxidant, anti-rejection activities. In P. cocos, the terpenoid backbone is biosynthesized via mevalonate pathway(MVA pathway) and produce various bioactive terpenoids in further steps. Phosphomevalonate kinase catalyzes a reversible reaction between mevalonate 5-phosphate(MVAP) and mevalonate 5-diphosphate in the MVA pathway, this is the rate-limiting step during the conversion of mevalonate to squalene. Its gene is expressed at relatively low level and regulated by many terpenoid products, thus it is vital for regulation of the whole terpenoids producing pathway. In this study, phosphomevalonate kinase gene from P. cocos(PcPMK) was cloned, the gene function was identified with yeast complementation assay and in vitro enzymatic assay. The kinetic of the enzyme was further studied. The results were showed as follow in details:1 Cloned PcPMK gene The 1662 bp mRNA sequence with complete ORF and 3249 bp genomic DNA sequence of PcPMK gene, which contains 6 introns and 7 exons, were obtained. The gene promoter region was predicted containing many transcription factor binding sites for regulatory elements such as ABA, auxin and MYB. The encoding protein named PcPMK of 511 amino acids was predicted to be an unstable hydrophobic protein with calculated molecular weight 54.8kDa. PcPMK has a relatively higher similarity with PMK proteins from fungi. The phylogenetic analysis indicated that the clusters of proteins coordinated with the taxonomic lineage.Three enzyme activity related conserved motifs, like P-loop, which are characteristic of the GHMP protein family, were identified in the protein sequence, and these motifs were modeled to be adjacent at 3D structure level.2 PcPMK is complementary to yeast gene ERG8 The yeast expression plasmid vector pYES2-pcpmk was constructed and transformed into ERG8 heterozygous mutant yeast strain. The optimized protocol was followed for sporulation and digestion of asci, the transformed strain was incubated on McClary medium, 25℃ for 15 days, a mixture of vegetative cells and asci was then treated with 30mg/mL snailase at 30℃ for 60 min, followed by tetrad dissection. The desired haploid yeast strains were selected accoding to phenotypes and PCR mating type assay. The strains were quantitatively inoculated onto five plates of different media to display the results of gene complementation. The expected results were obtained, and that demonstrated that the cloned PcPMK gene possesses the activity of PMK genes.3 The recombinant PcPMK has the function of phosphomevalonate kinase The prokaryotic expression plasmid vector pCold-pcpmk was constructed and transformed into E. coli strain BL21(DE3). PcPMK protein, which expressed and purified using the optimized protocols, mixed with substrates MVAP and ATP for in vitro enzymatic assay. The expected products mevalonate 5-pyrophosphate and ADP were detected using UPLCMS in the assay mixture. The result demonstrated that the PcPMK recombinant protein possesses the catalytic function of ATP:5-phosphomevalonate phosphotransferase.4 PcPMK is Mn2+ denpendent enzyme The further enzyme kinetics studies indicated that PcPMK is Mn2+ dependent, Mg2+ could also partially activate its activity. The PcPMK is sensitive to ATP concentration and inhibited by ATP under high ATP concentration. The activity of PcPMK was measured using a microplate continuous enzyme assay in an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase. The Km value and Vmax value for ATP were determined to be 67.7±7.5μmol/L and 6.21±0.19μmol/min/mg protein, respectively, and the values of 104.3±9.8μmol/L, 6.75±0.18μmol/min/mg protein for MVAP.In all, this study cloned PcPMK gene for the fist time, its function validated with yeast complementation assay and in vitro enzymatic assay. The gene sequence and protein sequence were systematically analyzed using a variety of bioinformatics tools. The protein 3D structure was modeled and analyzed. The Mn2+ dependence of PcPMK was demonstrated, and the kinetic parameters to MVAP and ATP were determined.