Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum

Abstract

Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts.

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis

“…Similar “metabolons”, defined as “supramolecular complexes of sequential metabolic enzymes and cellular structural elements” [119], have been observed in other systems, such as the tricarboxylic acid cycle [120], the purinosome [121], dhurrin biosynthesis [122], and steroid biosynthesis [123]. Identifying the functions of such metabolon organization remains an active area of research with multiple hypotheses.…”

Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers

“…This complex is formed by a soluble glycotransferase and three membrane-anchored proteins along with associated lipids. Treatment of microsome membranes with SMA2000 transfers the entire endogenous complex into SMALPs with 10–25 nm diameters and yields of 80%, while conventional detergents such as cholate dissociate the complex [33]. The soluble subunit can be added to the transmembrane assembly in the SMALP to modulate the activity of the transmembrane components.…”

A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid α-Onocerin

“…The preparation of mRFP1 and SbCYP98A1-mRFP1 constructs was as described (Laursen et al, 2016). The construction of OsONS1, OsSQE1, and OsSQE2 C-terminally fused to eGFP or mRFP1 was done by amplifying the fulllength coding sequences with appropriate primers (Supplemental Table S1), and the amplicons were inserted in the pCAMBIA1300/UeGFP or pCAM-BIA1300/UmRFP1 vector, respectively, by the single-insert USER cloning technique (Geu-Flores et al, 2007).…”

“…The construction of OsONS1, OsSQE1, and OsSQE2 C-terminally fused to eGFP or mRFP1 was done by amplifying the fulllength coding sequences with appropriate primers (Supplemental Table S1), and the amplicons were inserted in the pCAMBIA1300/UeGFP or pCAM-BIA1300/UmRFP1 vector, respectively, by the single-insert USER cloning technique (Geu-Flores et al, 2007). Finally, to create the OsONS1 construct N-terminally fused to eGFP, the full-length coding sequence of OsONS1 was amplified with appropriate primers and the amplicons were inserted into the pCAMBIA1300/eGFPU vector (Laursen et al, 2016) with USER cloning. For transient expression in N. benthamiana, constructs were transformed to A. tumefaciens LBA4404 virGN54D (van der Fits et al, 2000).…”

“…An SP5x confocal laser scanning microscopy device equipped with a DM6000 microscope (Leica) was used to record images of enzyme subcellular localization with settings described by Laursen et al (2016). FLIM was performed by time-correlated single-photon counting using the Microtime 200 system (Picoquant), and measurements were done using an Olympus IX70 microscope with pulse laser excitation at 485 nm provided by a Picosecond Diode Laser (PDL 828 SepiaII; Picoquant) with settings as described (Laursen et al, 2016). Samples were scanned for 232 s with the accurate FLIM method on the SymPhoTime64 software (Picoquant).…”