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Plant and Cell Physiology Advance Access published online on January 19, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci021
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Plant and Cell Physiology 2005 © The Japanese Society of Plant Physiologists (JSPP); all rights researved.
Received October 12, 2004
Accepted November 10, 2004

Regular Paper

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

Taketo Okada 1, Masami Yokota Hirai 2, Hideyuki Suzuki 1, Mami Yamazaki 1, and Kazuki Saito 2*

1 Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan
2 Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan; CREST of Japan Science and Technology Agency (JST), Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan

* To whom correspondence should be addressed.
Kazuki Saito, E-mail: ksaito{at}faculty.chiba-u.jp


  Abstract

A novel acyltransferase committed to the final step of quinolizidine alkaloid biosynthesis, tigloyl-CoA:(-)-13{alpha}-hydroxymultiflorine/(+)-13{alpha}-hydroxylupanine O-tigloxyltransferase, has been purified from Lupinus albus. The internal amino acid sequences were determined with protease-digested fragments of 25 kDa 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 deduced amino acid sequences indicated that this alkaloid acyltransferase belongs to a unique sub-family 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 (-)-13{alpha}-hydroxymultiflorine and (+)-13{alpha}-hydroxylupanine. Benzoyl-CoA could also efficiently serve 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 the nature, is committed to control the quinolizidine alkaloid patterns in a tissue-specific manner.

Keywords: Acyltransferase; Alkaloid; HMT/HLT; Lupinus albus; Tigloyltransferase.
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