Metabolon formation in dhurrin biosynthesis.
Synthesis of the tyrosine derived cyanogenic glucoside dhurrin in Sorghum bicolor is catalyzed by two multifunctional, membrane bound cytochromes P450, CYP79A1 and CYP71E1, and a soluble UDPG-glucosyltransferase, UGT85B1 (Tattersall, D.B., Bak, S., Jones, P.R., Olsen, C.E., Nielsen, J.K., Hansen, M.L., Høj, P.B., Møller, B.L., 2001. Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293, 1826-1828). All three enzymes retained enzymatic activity when expressed as fluorescent fusion proteins in planta. Transgenic Arabidopsis thaliana plants that produced dhurrin were obtained by co-expression of CYP79A1/CYP71E1-CFP/UGT85B1-YFP and of CYP79A1/CYP71E1/UGT85B1-YFP but not by co-expression of CYP79A1-YFP/CYP71E-CFP/UGT85B1. The lack of dhurrin formation upon co-expression of the two cytochromes P450 as fusion proteins indicated that tight interaction was necessary for efficient substrate channelling. Transient expression in S. bicolor epidermal cells as monitored by confocal laser scanning microscopy showed that UGT85B1-YFP accumulated in the cytoplasm in the absence of CYP79A1 or CYP71E1. In the presence of CYP79A1 and CYP71E1, the localization of UGT85B1 shifted towards the surface of the ER membrane in the periphery of biosynthetic active cells, demonstrating in planta dhurrin metabolon formation.
Nielsen
Kirsten Annette
KA
Plant Biochemistry Laboratory, Department of Plant Biology, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Tattersall
David B
DB
Jones
Patrik Raymond
PR
Møller
Birger Lindberg
BL
D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D045868:Sorghum / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*
D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D009570:Nitriles; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D005964:Glucosyltransferases; C011220:dhurrin