Publications

• Molecular characterization of a novel quinolizidine alkaloid O-tigloyltransferase: cDNA cloning, catalytic activity of recombinant protein and expression analysis in Lupinus plants.

  Okada T., Hirai M.Y., Suzuki H., Yamazaki M., Saito K.

  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 f ... >> More

  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. << Less

  Plant Cell Physiol. 46:233-244(2005) [PubMed] [EuropePMC]

• A novel O-tigloyltransferase for alkaloid biosynthesis in plants. Purification, characterization, and distribution in Lupinus plants.

  Suzuki H., Murakoshi I., Saito K.

  A novel acyltransferase for alkaloid metabolism, tigloyl-CoA: (-)-13 alpha-hydroxymultiflorine/(+)-13 alpha-hydroxylupanine O-tigloyltransferase (HMT/HLTase), a monomeric 50-kDa protein, was purified to homogeneity from 10-day-old Lupinus termis seedlings. There were two isoforms of this acyltrans ... >> More

  A novel acyltransferase for alkaloid metabolism, tigloyl-CoA: (-)-13 alpha-hydroxymultiflorine/(+)-13 alpha-hydroxylupanine O-tigloyltransferase (HMT/HLTase), a monomeric 50-kDa protein, was purified to homogeneity from 10-day-old Lupinus termis seedlings. There were two isoforms of this acyltransferase with the same molecular mass (50 kDa) but slightly different isoelectric points (pI 7.8 and 7.6). These two isoforms showed the same catalytic activity of tigloyl transfer from tigloyl-CoA to (-)-13 alpha-hydroxymultiflorine and (+)-13 alpha-hydroxylupanine, which belong to the same (7S, 9S) enantiomeric series of tetracyclic quinolizidine alkaloids; whereas no activity was detected toward an antipodal (7R, 9R) alkaloid, (-)-baptifoline, or to bicyclic quinolizidine alkaloids, (+)-epilupinine and (-)-lupinine. The Km values for HMTase activity were determined to be 21 microM and 46 microM for (-)-13 alpha-hydroxymultiflorine and tigloyl-CoA, respectively; and for HLTase activity, 27 microM and 52 microM for (+)-13 alpha-hydroxylupanine and tigloyl-CoA, respectively. The activity was inhibited by CoASH in a competitive manner, and by (+)-lupanine and (+)-epilupinine in a partially noncompetitive manner. The enzyme showed the highest activity around pH 8.0 and was inactivated by heat treatment and by the addition of sulfhydryl blocking reagents. Such tigloyltransferases for quinolizidine alkaloid metabolism are distributed in some Lupinus species and Cytisus scoparius, in which tigloyl alkaloids are accumulated in addition to non-ester-type alkaloids, but not in other lupin plants, in which only non-ester-type alkaloids are present. << Less

  J. Biol. Chem. 269:15853-15860(1994) [PubMed] [EuropePMC]