Molecular characterization of a novel quinolizidine alkaloid O-tigloyltransferase: cDNA cloning, catalytic activity of recombinant protein and expression analysis in Lupinus plants.
A novel acyltransferase committed to the final step of quinolizidine alkaloid biosynthesis, tigloyl-CoA:(-)-13alpha-hydroxymultiflorine/(+)-13alpha-hydroxylupanine O-tigloyltransferase, has been purified from Lupinus albus. The internal amino acid sequences were determined with protease-digested fragments of 25 and 30 kDa bands, allowing design of primers for amplification of cDNA fragments by polymerase chain reaction. Using an amplified fragment as the probe, a full-length cDNA clone was isolated. Sequence analysis revealed that the cDNA encodes a protein of 453 amino acids with a molecular mass of 51.2 kDa. Phylogenetic analysis of the deduced amino acid sequences indicated that this alkaloid acyltransferase belongs to a unique subfamily of a plant acyl-CoA-dependent acyltransferase gene family. The cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity purified and cleaved to yield the recombinant enzyme for the study of catalytic properties. The recombinant enzyme catalyzed the acyltransfer reaction from tigloyl-CoA to (-)-13alpha-hydroxymultiflorine and (+)-13alpha-hydroxylupanine. Benzoyl-CoA could also serve efficiently as an acyl donor for these hydroxylated alkaloids. RNA blot analysis suggested that the gene was expressed in roots and hypocotyls but not in cotyledons and leaves. These results indicated that this specialized acyltransferase, isolated for the first time as tigloyltransferase from nature, is committed to control the quinolizidine alkaloid patterns in a tissue-specific manner.
Okada
Taketo
T
Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
Hirai
Masami Yokota
MY
Suzuki
Hideyuki
H
Yamazaki
Mami
M
Saito
Kazuki
K
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000470:Alkaloids / Q000096:biosynthesis; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression; D031297:Lupinus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000470:Alkaloids; D018076:DNA, Complementary; D018744:DNA, Plant; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; D000217:Acyltransferases; C087441:tigloyl-CoA-13-hydroxymultiflorine-13-hydroxyupanine O-tigloyltransferase