The binding of UDP-glucosyltransferase to the cytochrome P450s in dhurrin biosynthesis

TY - CONF

T1 - The binding of UDP-glucosyltransferase to the cytochrome P450s in dhurrin biosynthesis

AU - Baden, Camilla Knudsen

AU - Laursen, Tomas

AU - Kannangara, Rubini Maya

AU - Bertram, Nicolas

AU - Cardenas Gomez, Marite

AU - Møller, Birger Lindberg

N1 - Poster presented at the Plant Biotech Denmark meeting

PY - 2013

Y1 - 2013

N2 - Cyanogenic glucosides are amino-acid derived plant chemical defense compounds against generalist herbivores. The subtropical crop plant Sorghum bicolor synthesizes the L-tyrosine derived cyanogenic glucoside dhurrin through the activity of two multifunctional cytochrome P450s (CYPs), CYP79A1 and CYP71E1 and an UDP-glucosyltransferase UGT85B1. The two CYPs are dependent on electrons donated from the cytochrome P450 oxido reductase (POR) for activity. Upon tissue disruption dhurrin is deglucosylated to an α-hydroxynitrile by specific β-glucosidases. The α-hydroxynitrile is unstable and will dissociate into hydrogen cyanide and a keto compound. The biosynthesis of dhurrin is highly channeled as only trace amounts of intermediates are detected in planta, therefore it is thought that the biosynthetic enzymes form transient enzyme complexes, metabolons (1, 2). The CYP79A1, CYP71E1 and POR are all ER localized by an N-terminal anchor, whereas the UGT85B1 is thought to be cytosolic with tight association to the ER. Fluorescence resonance energy transfer (FRET) studies have shown that the soluble UGT is associated close to the ER localized CYPs, which enables efficient glycosylation and stabilization of the labile α-hydroxynitrile produced by the CYPs (3). Protein homology modeling has revealed that a hydrophobic patch in the loop B region of the sorghum UGT85B1 might be involved in the interaction with the membrane-bound CYPs. This hydrophobic patch could play a role in the affinity between the UGT and the CYPs or the lipid environment of the ER membrane (4). This project aims to understand the role of loop B in the coupling of the UGT with the dhurrin metabolon. Different UGT constructs have been made with alternative versions of the loop B region to investigate the importance of the loop structure for mediating interaction. Seven constructs have been made in total to highlight whether or not loop B is the site of interaction between the cytosolic UGT and the membrane-bound CYPs. These UGT versions are analyzed by coupled activity assays with the CYPs from microsome preparations or purified liposome reconstituted enzymes. Furthermore, interactions between CYP79A1 or CYP71E1 reconstituted in nanoscale lipid bilayer discs, nanodiscs, with the different UGT versions are analyzed by quartz crystal microbalance (QCM). Upon formation of a nanodisc monolayer at the quartz crystal, containing either CYP79A1 or CYP71E1, the binding of the UGT is monitored by a shift in the density of the immobilized layer. We believe that these studies will provide a detailed understanding of the formation of metabolons in the biosynthesis of plant natural products. Furthermore, the synthetic biology approach might enable the modification of UGTs in order to design desirable glycosylation patterns of bio-active compounds. 1) Jensen, K., Osmani, S., Hamann, T., Naur, P., & Møller, B.L. 2011. Homology modeling of the three membrane proteins of the dhurrin metabolon: Catalytic sites, membrane surface association and protein-protein interactions. Phytochemistry, 72(17):2113–2123 2) Jørgensen, K., Rasmussen, A.V., Morant, M., Nielsen, A.H., Bjarnholt, N., Zagrobelny, M., Bak, S., Møller, B.L., 2005. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr. Opin. Plant Biol. 8, 280–291. 3) Nielsen, K.A., Tattersall, D.B., Jones, P.R., Møller, B.L., 2008. Metabolon formation in dhurrin biosynthesis. Phytochemistry 69, 88–98. 4) Thorsøe, K.S., Bak, S., Olsen, C.E., Imberty, A., Breton, C., Møller, B.L., 2005. Determination of catalytic key amino acids and UDP sugar donor specificity of the cyanohydrin glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular modeling substantiated by site-specific mutagenesis and biochemical analyses. Plant Physiol. 139, 664–673.

AB - Cyanogenic glucosides are amino-acid derived plant chemical defense compounds against generalist herbivores. The subtropical crop plant Sorghum bicolor synthesizes the L-tyrosine derived cyanogenic glucoside dhurrin through the activity of two multifunctional cytochrome P450s (CYPs), CYP79A1 and CYP71E1 and an UDP-glucosyltransferase UGT85B1. The two CYPs are dependent on electrons donated from the cytochrome P450 oxido reductase (POR) for activity. Upon tissue disruption dhurrin is deglucosylated to an α-hydroxynitrile by specific β-glucosidases. The α-hydroxynitrile is unstable and will dissociate into hydrogen cyanide and a keto compound. The biosynthesis of dhurrin is highly channeled as only trace amounts of intermediates are detected in planta, therefore it is thought that the biosynthetic enzymes form transient enzyme complexes, metabolons (1, 2). The CYP79A1, CYP71E1 and POR are all ER localized by an N-terminal anchor, whereas the UGT85B1 is thought to be cytosolic with tight association to the ER. Fluorescence resonance energy transfer (FRET) studies have shown that the soluble UGT is associated close to the ER localized CYPs, which enables efficient glycosylation and stabilization of the labile α-hydroxynitrile produced by the CYPs (3). Protein homology modeling has revealed that a hydrophobic patch in the loop B region of the sorghum UGT85B1 might be involved in the interaction with the membrane-bound CYPs. This hydrophobic patch could play a role in the affinity between the UGT and the CYPs or the lipid environment of the ER membrane (4). This project aims to understand the role of loop B in the coupling of the UGT with the dhurrin metabolon. Different UGT constructs have been made with alternative versions of the loop B region to investigate the importance of the loop structure for mediating interaction. Seven constructs have been made in total to highlight whether or not loop B is the site of interaction between the cytosolic UGT and the membrane-bound CYPs. These UGT versions are analyzed by coupled activity assays with the CYPs from microsome preparations or purified liposome reconstituted enzymes. Furthermore, interactions between CYP79A1 or CYP71E1 reconstituted in nanoscale lipid bilayer discs, nanodiscs, with the different UGT versions are analyzed by quartz crystal microbalance (QCM). Upon formation of a nanodisc monolayer at the quartz crystal, containing either CYP79A1 or CYP71E1, the binding of the UGT is monitored by a shift in the density of the immobilized layer. We believe that these studies will provide a detailed understanding of the formation of metabolons in the biosynthesis of plant natural products. Furthermore, the synthetic biology approach might enable the modification of UGTs in order to design desirable glycosylation patterns of bio-active compounds. 1) Jensen, K., Osmani, S., Hamann, T., Naur, P., & Møller, B.L. 2011. Homology modeling of the three membrane proteins of the dhurrin metabolon: Catalytic sites, membrane surface association and protein-protein interactions. Phytochemistry, 72(17):2113–2123 2) Jørgensen, K., Rasmussen, A.V., Morant, M., Nielsen, A.H., Bjarnholt, N., Zagrobelny, M., Bak, S., Møller, B.L., 2005. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr. Opin. Plant Biol. 8, 280–291. 3) Nielsen, K.A., Tattersall, D.B., Jones, P.R., Møller, B.L., 2008. Metabolon formation in dhurrin biosynthesis. Phytochemistry 69, 88–98. 4) Thorsøe, K.S., Bak, S., Olsen, C.E., Imberty, A., Breton, C., Møller, B.L., 2005. Determination of catalytic key amino acids and UDP sugar donor specificity of the cyanohydrin glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular modeling substantiated by site-specific mutagenesis and biochemical analyses. Plant Physiol. 139, 664–673.

KW - Faculty of Science

KW - Synthetic Biology

KW - plant biochemistry

M3 - Poster

ER -