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.

SummaryProkaryotic toxin-antitoxin (TA) loci consist of two genes in an operon that encodes a metabolically stable toxin and an unstable antitoxin. The antitoxin neutralizes its cognate toxin by forming a tight complex with it. In all cases known, the antitoxin autoregulates TA operon transcription by binding to one or more operators in the promoter region while the toxin functions as a co-repressor of transcription. Interestingly, the toxin can also stimulate TA operon transcription. Here we analyse mechanistic aspects of how RelE of Escherichia coli can function both as a co-repressor and as a derepressor of relBE transcription. When RelB was in excess to RelE, two trimeric RelB 2•RelE complexes bound cooperatively to two adjacent operator sites in the relBE promoter region and repressed transcription. In contrast, RelE in excess stimulated relBE transcription and released the RelB2•RelE complex from operator DNA. A mutational analysis of the operator sites showed that RelE in excess counteracted cooperative binding of the RelB2•RelE complexes to the operator sites. Thus, RelE controls relBE transcription by conditional cooperativity.

RelB, the ribbon–helix–helix (RHH) repressor encoded by the relBE toxin–antitoxin locus of Escherichia coli, interacts with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE; that is, RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate, and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes that map to important amino acid positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA binding of a subset of these mutant RHH proteins by gel-shift assays, transcriptional fusion assays and a structure model of RelB–DNA revealed amino acid residues making crucial DNA–backbone contacts within the operator (relO) DNA. Mutational and footprinting analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6-bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon degraded RelB in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo.

SummaryTrehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.

For more than 420 million years, plants, insects and their predators have co-evolved based on a chemical arms race including deployment of refined chemical defence systems by each player. Cyanogenic glucosides are produced by numerous plants and by some specialized insects and serve an important role as defence compounds in these intimate interactions. Burnet moth larvae are able to sequester cyanogenic glucosides from their food plant as well as to carry out de novo biosynthesis. Here we show that three genes (CYP405A2, CYP332A3 and UGT33A1) encode the entire biosynthetic pathway of cyanogenic glucosides in the Burnet moth Zygaena filipendulae. In both plants and insects, convergent evolution has led to two multifunctional P450 enzymes each catalysing unusual reactions and a glucosyl-transferase acting in sequence to catalyse cyanogenic glucoside formation. Thus, plants and insects have independently found a way to package a cyanide time bomb to fend off herbivores and predators.

A major challenge in the research on membrane-anchored and integral membrane protein complexes is to obtain these in a functionally active, water-soluble, and monodisperse form. This requires the incorporation of the membrane proteins into a native-like membrane or detergent micelle that mimics the properties of the original biological membrane. However, solubilization in detergents or reconstitution in liposomes or supported monolayers sometimes suffers from loss of activity and problematic analyses due to heterogeneity and aggregation. A developing technology termed nanodiscs exploits discoidal phospholipid bilayers encircled by a stabilizing amphipatic helical membrane scaffold protein to reconstitute membranes with integral proteins. After reconstitution, the membrane nanodisc is soluble, stable, and monodisperse. In the present review, we outline the biological inspiration for nanodiscs as discoidal high-density lipoproteins, the assembly and handling of nanodiscs, and finally their diverse biochemical applications. In our view, major advantages of nanodisc technology for integral membrane proteins is homogeneity, control of oligomerization state, access to both sides of the membrane, and control of lipids in the local membrane environment of the integral protein.

Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

Nanodiscs are self-assembled soluble discoidal phospholipids bilayers encirculated by an amphipathic protein that together provide a functional stabilized membrane disk for the incorporation of membrane-bound and membrane-associated molecules. The scope of the present work is to investigate how nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. Cholera toxin and its glycolipid receptor G(M1) constitute a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodiscs containing the glycolipid receptor G(M1) in lipid bilayers, enabling measurements of binding of its soluble interaction partner cholera toxin B subunit to the receptor with the sensorchip-based surface plasmon resonance (SPR) technology. The measured stoichiometric and kinetic values of the interaction are in agreement with those reported by previous studies, thus providing proof-of-principle that nanodiscs can be employed for kinetic SPR studies.