Journal Article

Molecular Characterization of a Novel Quinolizidine Alkaloid <i>O</i>-Tigloyltransferase: cDNA Cloning, Catalytic Activity of Recombinant Protein and Expression Analysis in <i>Lupinus</i> Plants

Taketo Okada, Masami Yokota Hirai, Hideyuki Suzuki, Mami Yamazaki and Kazuki Saito

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 1, pages 233-244
Published in print January 2005 | ISSN: 0032-0781
Published online January 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci021
Molecular Characterization of a Novel Quinolizidine Alkaloid O-Tigloyltransferase: cDNA Cloning, Catalytic Activity of Recombinant Protein and Expression Analysis in Lupinus Plants

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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A novel acyltransferase committed to the final step of quinolizidine alkaloid biosynthesis, tigloyl-CoA:(–)-13α-hydroxymultiflorine/(+)-13α-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 (–)-13α-hydroxymultiflorine and (+)-13α-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.

Keywords: Keywords: Acyltransferase — Alkaloid — HMT/HLT — Lupinus albus — Tigloyltransferase.; Abbreviations: DEPC, diethylpyrocarbonate; GST, glutathione S-transferase; HMT/HLT, tigloyl-CoA:(–)-13α-hydroxymultiflorine/ (+)-13α-hydroxylupanine O-tigloyltransferase; LC-PDA-ESI/MS, liquid chromatography-photodiode array detection-electrospray ionization-mass spectrometry; ORF, open reading frame; RACE, rapid amplification of cDNA end; RT–PCR, reverse transcription–polymerase chain reaction.

Journal Article.  7139 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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