Expanding the Toolbox for Bicelle-Forming Surfactant-Lipid Mixtures.

Bicelles are disk-shaped models of cellular membranes used to study lipid-protein interactions, as well as for structural and functional studies on transmembrane proteins. One challenge for the incorporation of transmembrane proteins in bicelles is the limited range of detergent and lipid combinations available for the successful reconstitution of proteins in model membranes. This is important, as the function and stability of transmembrane proteins are very closely linked to the detergents used for their purification and to the lipids that the proteins are embedded in. Here, we expand the toolkit of lipid and detergent combinations that allow the formation of stable bicelles. We use a combination of dynamic light scattering, small-angle X-ray scattering and cryogenic electron microscopy to perform a systematic sample characterization, thus providing a set of conditions under which bicelles can be successfully formed.

Anthocyanic Vacuolar Inclusions: From Biosynthesis to Storage and Possible Applications.

The ability of plants to accumulate specific metabolites in concentrations beyond their solubility in both aqueous and lipid environments remains a key question in plant biology. Natural Deep Eutectic Solvents (NADES) are mixtures of natural compounds in specific molar ratios, which interact through hydrogen bonding. This results in a viscous liquid that can solubilize high amounts of natural products while maintaining a negligible vapor pressure to prevent release of volatile compounds. While all the components are presents in plant cells, identifying experimental evidence for the occurrence of NADES phases remains a challenging quest. Accumulation of anthocyanin flavonoids in highly concentrated inclusions have been speculated to involve NADES as an inert solvent. The inherent pigment properties of anthocyanins provide an ideal system for studying the formation of NADES in a cellular environment. In this mini-review we discuss the biosynthesis of modified anthocyanins that facilitate their organization in condensates, their transport and storage as a specific type of phase separated inclusions in the vacuole, and the presence of NADES constituents as a natural solution for storing high amounts of flavonoids and other natural products. Finally, we highlight how the knowledge gathered from studying the discussed processes could be used for specific applications within synthetic biology to utilize NADES derived compartments for the production of valuable compounds where the production is challenged by poor solubility, toxic intermediates or unstable and volatile products.

Structure-guided engineering of key amino acids in UGT85B1 controlling substrate and stereo-specificity in aromatic cyanogenic glucoside biosynthesis.

Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure-function relationships controlling scope and specificity remain poorly understood. Here, we report substrate-bound crystal structures and rational engineering of substrate and stereo-specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)-p-hydroxymandelonitrile to (S)-mandelonitrile by combining a mutation to abolish hydrogen bonding to the p-hydroxyl group with a mutation to provide steric hindrance at the p-hydroxyl group binding site (V132A/Q225W). Further, stereo-specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo-specificities in the cyanogenic glucoside biosynthetic UGTs. The structure-guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications.

Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts.

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-ß-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.

Membrane anchoring facilitates colocalization of enzymes in plant cytochrome P450 redox systems.

Plant metabolism depends on cascade reactions mediated by dynamic enzyme assemblies known as metabolons. In this context, the cytochrome P450 (P450) superfamily catalyze key reactions underpinning the unique diversity of bioactive compounds. In contrast to their soluble bacterial counterparts, eukaryotic P450s are anchored to the endoplasmic reticulum membrane and serve as metabolon nucleation sites. Hence, membrane anchoring appears to play a pivotal role in the evolution of complex biosynthetic pathways. Here, a model membrane assay enabled characterization of membrane anchor dynamics by single molecule microscopy. As a model system, we reconstituted the membrane anchor of cytochrome P450 oxidoreductase (POR), the ubiquitous electron donor to all microsomal P450s. The transmembrane segment in the membrane anchor of POR is relatively conserved, corroborating its functional importance. We observe dynamic colocalization of the POR anchors in our assay suggesting that membrane anchoring might promote intermolecular interactions and in this way impact assembly of metabolic multienzyme complexes.

Biased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase.

Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.

Stabilization of dhurrin biosynthetic enzymes from Sorghum bicolor using a natural deep eutectic solvent.

In recent years, ionic liquids and deep eutectic solvents (DESs) have gained increasing attention due to their ability to extract and solubilize metabolites and biopolymers in quantities far beyond their solubility in oil and water. The hypothesis that naturally occurring metabolites are able to form a natural deep eutectic solvent (NADES), thereby constituting a third intracellular phase in addition to the aqueous and lipid phases, has prompted researchers to study the role of NADES in living systems. As an excellent solvent for specialized metabolites, formation of NADES in response to dehydration of plant cells could provide an appropriate environment for the functional storage of enzymes during drought. Using the enzymes catalyzing the biosynthesis of the defense compound dhurrin as an experimental model system, we demonstrate that enzymes involved in this pathway exhibit increased stability in NADES compared with aqueous buffer solutions, and that enzyme activity is restored upon rehydration. Inspired by nature, application of NADES provides a biotechnological approach for long-term storage of entire biosynthetic pathways including membrane-anchored enzymes.

Molecular snapshots of dynamic membrane-bound metabolons.

Numerous biosynthetic pathways have been shown to assemble at the surface of cellular membranes into efficient dynamic supramolecular assemblies termed metabolons. In response to environmental stimuli, metabolons assemble on-demand making them highly dynamic and fragile. This transient nature has previously hampered isolation and molecular characterization of dynamic metabolons. In contrast to conventional detergents, which tend to disrupt weak protein-protein interactions and remove lipids, the competence of a styrene maleic acid copolymer to carve out discrete lipid nanodisc from membranes offers immense potential for isolation of intact protein assemblies. Here, we present a method to extract the entire membrane-bound dhurrin pathway directly from microsomal fractions of the cereal Sorghum bicolor. This detergent-free nanodisc approach may be generally transposed for isolation of entire plant biosynthetic metabolons. This method provides a simple practical toolkit for the study of membrane protein complexes.

Dynamic metabolic solutions to the sessile life style of plants.

Covering: up to 2018 Plants are sessile organisms. To compensate for not being able to escape when challenged by unfavorable growth conditions, pests or herbivores, plants have perfected their metabolic plasticity by having developed the capacity for on demand synthesis of a plethora of phytochemicals to specifically respond to the challenges arising during plant ontogeny. Key steps in the biosynthesis of phytochemicals are catalyzed by membrane-bound cytochrome P450 enzymes which in plants constitute a superfamily. In planta, the P450s may be organized in dynamic enzyme clusters (metabolons) and the genes encoding the P450s and other enzymes in a specific pathway may be clustered. Metabolon formation facilitates transfer of substrates between sequential enzymes and therefore enables the plant to channel the flux of general metabolites towards biosynthesis of specific phytochemicals. In the plant cell, compartmentalization of the operation of specific biosynthetic pathways in specialized plastids serves to avoid undesired metabolic cross-talk and offers distinct storage sites for molar concentrations of specific phytochemicals. Liquid-liquid phase separation may lead to formation of dense biomolecular condensates within the cytoplasm or vacuole allowing swift activation of the stored phytochemicals as required upon pest or herbivore attack. The molecular grid behind plant plasticity offers an endless reservoir of functional modules, which may be utilized as a synthetic biology tool-box for engineering of novel biological systems based on rational design principles. In this review, we highlight some of the concepts used by plants to coordinate biosynthesis and storage of phytochemicals.

The Three Members of the Arabidopsis Glycosyltransferase Family 92 are Functional ß-1,4-Galactan Synthases.

Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched ß-1, 4-galactan. Plants in which all three genes were inactivated had no detectable ß-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.

Direct observation of multiple conformational states in Cytochrome P450 oxidoreductase and their modulation by membrane environment and ionic strength.

Cytochrome P450 oxidoreductase (POR) is the primary electron donor in eukaryotic cytochrome P450 (CYP) containing systems. A wealth of ensemble biophysical studies of Cytochrome P450 oxidoreductase (POR) has reported a binary model of the conformational equilibrium directing its catalytic efficiency and biomolecular recognition. In this study, full length POR from the crop plant Sorghum bicolor was site-specifically labeled with Cy3 (donor) and Cy5 (acceptor) fluorophores and reconstituted in nanodiscs. Our single molecule fluorescence resonance energy transfer (smFRET) burst analyses of POR allowed the direct observation and quantification of at least three dominant conformational sub-populations, their distribution and occupancies. Moreover, the state occupancies were remodeled significantly by ionic strength and the nature of reconstitution environment, i.e. phospholipid bilayers (nanodiscs) composed of different lipid head group charges vs. detergent micelles. The existence of conformational heterogeneity in POR may mediate selective activation of multiple downstream electron acceptors and association in complexes in the ER membrane.

Bifunctional glycosyltransferases catalyze both extension and termination of pectic galactan oligosaccharides.

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side-chains of rhamnogalacturonan-I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP-Gal to growing ß-1,4-galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP-α-d-Gal and the transfer of an arabinopyranose from UDP-ß-l-Arap to galactan chains. The two substrates share a similar structure, but UDP-α-d-Gal is the preferred substrate, with a 10-fold higher affinity. Transfer of Arap to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage.

Controlling Styrene Maleic Acid Lipid Particles through RAFT.

The ability of styrene maleic acid copolymers to dissolve lipid membranes into nanosized lipid particles is a facile method of obtaining membrane proteins in solubilized lipid discs while conserving part of their native lipid environment. While the currently used copolymers can readily extract membrane proteins in native nanodiscs, their highly disperse composition is likely to influence the dispersity of the discs as well as the extraction efficiency. In this study, reversible addition-fragmentation chain transfer was used to control the polymer architecture and dispersity of molecular weights with a high-precision. Based on Monte Carlo simulations of the polymerizations, the monomer composition was predicted and allowed a structure-function analysis of the polymer architecture, in relation to their ability to assemble into lipid nanoparticles. We show that a higher degree of control of the polymer architecture generates more homogeneous samples. We hypothesize that low dispersity copolymers, with control of polymer architecture are an ideal framework for the rational design of polymers for customized isolation and characterization of integral membrane proteins in native lipid bilayer systems.

Assembly of Dynamic P450-Mediated Metabolons-Order Versus Chaos.

PURPOSE OF REVIEW: We provide an overview of the current knowledge on cytochrome P450-mediated metabolism organized as metabolons and factors that facilitate their stabilization. Essential parameters will be discussed including those that are commonly disregarded using the dhurrin metabolon from Sorghum bicolor as a case study. RECENT FINDINGS: Sessile plants control their metabolism to prioritize their resources between growth and development, or defense. This requires fine-tuned complex dynamic regulation of the metabolic networks involved. Within the recent years, numerous studies point to the formation of dynamic metabolons playing a major role in controlling the metabolic fluxes within such networks. SUMMARY: We propose that P450s and their partners interact and associate dynamically with POR, which acts as a charging station possibly in concert with Cytb5. Solvent environment, lipid composition, and non-catalytic proteins guide metabolon formation and thereby activity, which have important implications for synthetic biology approaches aiming to produce high-value specialized metabolites in heterologous hosts.

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

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.

Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase.

Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, "nanodiscs", and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and -300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s(-1). POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products.

Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing.

The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to a huge number of different cytochromes P450s (from 50 to several hundred within one plant). Within the eudicotyledons, PORs can be divided into two major clades, POR 1 and POR 2. Based on our own sequencing analysis and publicly available data, we have identified 45 PORs from the angiosperm order Apiales. These were subjected to a phylogenetic analysis along with 237 other publicly available (NCBI and oneKP) POR sequences found within the clade Asterids. Here, we show that the order Apiales only harbor members of the POR 2 clade, which are further divided into two distinct subclades. This is in contrast to most other eudicotyledon orders that have both POR 1 and POR 2. This suggests that through gene duplications and one gene deletion, Apiales only contain members of the POR 2 clade. Three POR 2 isoforms from Thapsia garganica L., Apiaceae, were all full-length in an Illumina root transcriptome dataset (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes.

Nanodisc Films for Membrane Protein Studies by Neutron Reflection: Effect of the Protein Scaffold Choice.

Nanodisc films are a promising approach to study the equilibrium conformation of membrane bound proteins in native-like environment. Here we compare nanodisc formation for NADPH-dependent cytochrome P450 oxidoreductase (POR) using two different scaffold proteins, MSP1D1 and MSP1E3D1. Despite the increased stability of POR loaded MSP1E3D1 based nanodiscs in comparison to MSP1D1 based nanodiscs, neutron reflection at the silicon-solution interface showed that POR loaded MSP1E3D1 based nanodisc films had poor surface coverage. This was the case, even when incubation was carried out under conditions that typically gave high coverage for empty nanodiscs. The low surface coverage affects the embedded POR coverage in the nanodisc film and limits the structural information that can be extracted from membrane bound proteins within them. Thus, nanodisc reconstitution on the smaller scaffold proteins is necessary for structural studies of membrane bound proteins in nanodisc films.

Plasticity of specialized metabolism as mediated by dynamic metabolons.

The formation of specialized metabolites enables plants to respond to biotic and abiotic stresses, but requires the sequential action of multiple enzymes. To facilitate swift production and to avoid leakage of potentially toxic and labile intermediates, many of the biosynthetic pathways are thought to organize in multienzyme clusters termed metabolons. Dynamic assembly and disassembly enable the plant to rapidly switch the product profile and thereby prioritize its resources. The lifetime of metabolons is largely unknown mainly due to technological limitations. This review focuses on the factors that facilitate and stimulate the dynamic assembly of metabolons, including microenvironments, noncatalytic proteins, and allosteric regulation. Understanding how plants organize carbon fluxes within their metabolic grids would enable targeted bioengineering of high-value specialized metabolites.

Single molecule activity measurements of cytochrome P450 oxidoreductase reveal the existence of two discrete functional states.

Electron transfer between membrane spanning oxidoreductase enzymes controls vital metabolic processes. Here we studied for the first time with single molecule resolution the function of P450 oxidoreductase (POR), the canonical membrane spanning activator of all microsomal cytochrome P450 enzymes. Measurements and statistical analysis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date has remained masked in bulk kinetics. To ensure that we measured the inherent behavior of POR, we reconstituted the full length POR in "native like" membrane patches, nanodiscs. Nanodisc reconstitution increased stability by ∼2-fold as compared to detergent solubilized POR and showed significantly increased activity at biologically relevant ionic strength conditions, highlighting the importance of studying POR function in a membrane environment. This assay paves the way for studying the function of additional membrane spanning oxidoreductases with single molecule resolution.