Metabolon formation in natural product synthesis as studied using nano disc technology

Danish title: Metabolon-dannelse i syntesen af dhurrin studeret ved brug af nano discs

 

Co-location of enzymes catalyzing consecutive steps in a biosynthetic pathway is thought of as a central feature of cell metabolism. Such supra-molecular multi-enzyme complexes are also termed metabolons. The molecular mechanisms governing metabolon formation remain elusive. Precise knowledge of the nature of interacting peptide domains that define metabolon formation in combination with molecular modelling would enable incorporation of such domains into other enzymes to induce the assembly of enzymes from other biosynthetic pathways into metabolons and thus facilitate channeling of intermediates into desired new products like drugs, fragrances, flavours and pigments. This would offer significant advantages for industrial production in microbes, in vitro cell cultures and plants. The experimental approach is based on reconstitution of the dhurrin metabolon in NANODISCs followed by BIA-MS analyses to identify interacting protein domains We have previously elucidated the pathway for the cyanogenic glucoside dhurrin in Sorghum bicolor and demonstrated that the pathway is catalyzed by two membrane-bound cytochrome P450s denoted CYP79A1 and CYP71E1 and by a soluble UDPG-glucosyl transferase, denoted UGT85B1. The corresponding three cDNA clones have been isolated. All three proteins were obtained in active form by heterologous expression in Escherichia coli. Using genetic engineering, the entire pathway for dhurin synthesis has been transferred from sorghum to Arabidopsis thaliana. The transgenic A. thaliana plants obtained accumulated high amounts of dhurrin (5-7% of dry weight matter). All three enzymes were also shown to be active when expressed as fusion proteins carrying variants of green fluorescent proteins. This offered the possibility to examine the subcellular distribution of the proteins in intact plant cells by confocal microscopy. Transgenic plants in which the two P450s were simultaneously expressed as fusion proteins, had severely reduced levels of dhurrin. Because each fusion protein was shown to be functionally active, it was proposed that the reduction in dhurrin accumulation reflected that the fusion partners caused a steric hindrance that prevented proper enzyme interaction and thus obliterated substrate channeling. Confocal microscopy demonstrated that the soluble UGT85B1 is located in the cytosol when expressed in A. thaliana. However, when co-expressed with CYP79A1 and CYP71E1, the glucosyltransferase localizes to distinct domains of the endoplasmatic reticulum. This is the first visual demonstration of metabolon formation using confocal microscopy.

 

The Nanodisc technology has been developed in Professor Stephen Sligars laboratory in Illinois, USA. Nanodiscs are self-assembled nanostructures composed of phospholipids encircled by a genetically engineered artificial membrane scaffold protein (MSP) that can provide a soluble discoidal bilayer 8-20 nm in diameter. The diameter of the nanodisc is determined by the structure of the membrane scaffold protein used and determines whether a single, two or more membrane proteins are inserted into each Nanodisc by a simple self-assembly process. The Nanodisc provides a native membrane environment that faithfully reproduces a native bilayer environment and thus allows membrane protein targets to be effectively solubilized in a native-like environment wherein they maintain their physiological function and retain a topology in relation to the membrane bilayer mimicking that found in host cellular structures. Experience with the first cytochrome P450 and other membrane bound proteins incorporated into Nanodiscs suggests that these nanoscale assemblies will be extremely robust to loss of enzymatic function.

 

The availability of Nanodiscs thus overcomes previously encountered severe hurdles in the quest of understanding the molecular basis of substrate-product channeling mediated by macromolecular interactions with soluble enzyme factors. Nanodiscs with defined stoichiometry between the inserted components can be purified in amounts sufficient for an extensive analysis of their interaction with soluble recombinant proteins or with still unpredicted interacting partners in vitro using biospecific interaction analysis-MS (BIA-MS), a new surface plasmon resonance technique coupled to mass spectrometry. 

 

Researchers involved: Thomas Hamann, Kirsten Annette Nielsen, Søren Bak, Birger Lindberg Møller

Foreign collaborators: Stephen Sligar and Steven Grimme, University of Illinois, Urbana, USA; Dany Werck, Laboratory of Functional Genomics of Plant Cytochromes P450, Department of Plant Metabolic Responses, IBMP-CNRS, Strasbourg, France

 

Financial support: KVL PhD stipend, Human Frontier Science Program, Danish National Research Foundation, Research Council for Technology and Production.