title: [Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata].
abstract: Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.
authors.forename: Ci
authors.initials: C
authors.lastname: Di
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Yuntao
authors.initials: Y
authors.lastname: Tang
authors.forename: Qingzhong
authors.initials: Q
authors.lastname: Peng
mesh_terms: D002262:Carboxy-Lyases / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D032501:Huperzia / Q000201:enzymology* / Q000235:genetics; D008239:Lysine / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C021520:lysine decarboxylase; D008239:Lysine