Abstract
Quinolizidine (Lupin) alkaloids (QAs) are a group of secondary compounds which are mainly found in the family Leguminosae. In Lupinus plant, tigloyl esters of alkaloids are found as major forms among ester-type alkaloids, which are produced by action of tigloyl-CoA:13α-hydroxymultiflorine/13α-hydroxylupanine O-tigloyltransferase (HMT/HLT). We report here, for the first time, molecular cloning and characterization of HMT/HLT. HMT/HLT has been purified from L. albus. The internal amino acid sequences were determined, allowing design of primers for RT-PCR. Using an amplified fragment as the probe, a full-length cDNA clone was isolated from cDNA library. The functional analysis was carried out with the recombinant gene product heterogeneously expressed in E. coli. We confirmed that the recombinant enzyme catalyzed the acyltransfer reaction from tigloyl-CoA to 13α-hydroxymultiflorine and 13α-hydroxylupanine. The substrate specificity, the assays with inhibitors and the nucleic acid hybridization analyses of HMT/HLT will be also presented.
