Abstract
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.
Highlights
A novealcyltransferaseforalkaloidmetabolism, gation at the purifiedenzyme level are presumably the low tigloyl-CoA:(-)-13a-hydroxymultiflorine/(+)-l3~-hy~oxl-evels of enzymatic activitiesfor the metabolic steps of interest, ylupanine 0-tigloyltransferase (HMTlHLTase), a mono- compared with those in primary metabolism, and the subsemeric SO-kDa protein, was purified to homogeneity froqmuent difficulty in purifying small amountsof enzyme proteins
13a-hydroxymultiflorineand (+)-13a-hydroxylupanine, which belong to the sam(7e5,9S)enantiomeric seriesof tetracyclic quinolizidine alkaloids; whereanso activity was detected toward an antipod(al7R,9R)alkaloid
P-coumaroyl- and feruloyl-CoA acted as acyl donors for neither tetracyclic alkaloids,13-OH-mu1and 13-OHlup, nor bicyclic alkaloids,(+I-epilupinine and (-)-lupinine, toward which the enzymaticactivities of p-coumaroyl- and feruloyl-transferases were detected in L. hirsutus [17]
Summary
All purification procedureswere performedat 4 "C except forSteps 7, 8, and 9,whichwere carried out with a HPLC system (HITACHI L-6210)at room temperature. Termis seedlings were homogenized with polyvinylpolypyrrolidone(290g)in 8,000ml of buffer A(200 mM potassium-Pi,pH 8.0, containing 10 m 2-mercaptoethanol,0.5 IIIM EDTAand 250 mM sucrose)with a Waring blenderT. The solution was desalted by passage through a Sephdex G-25 column(8.4 X 37.5 cm)pre-equilibrated with buffer B. Step 3: First Anion-exchangeChromatography on DEAE-Sepharose Fast Flow-The desalted eluate (750 ml) from Step 2 was chromatographed on a DEW-Sepharose Fast Flow column (3.4 x 22 cm) preequilibrated with buffer B. Afterwashing out the unadsorbed proteins with 400 ml of buffer B, the adsorbedproteins were eluted with a 800ml of buffer B containing 0.3 M NaC1. Step 4: Dye-Ligand AfFnity Chromatography on CibacronBlue 3GA-The unadsorbed protein solution (1,150 ml) from Step 3 was chromatographed on a Cibacron Blue 3GA column (3.4 x 15.5 cm)preequilibrated with buffer B.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
