Organ transformation by environmental disruption of protein integrity and epigenetic memory in Drosophila.

Despite significant progress in understanding epigenetic reprogramming of cells, the mechanistic basis of "organ reprogramming" by (epi-)gene-environment interactions remained largely obscure. Here, we use the ether-induced haltere-to-wing transformations in Drosophila as a model for epigenetic "reprogramming" at the whole organism level. Our findings support a mechanistic chain of events explaining why and how brief embryonic exposure to ether leads to haltere-to-wing transformations manifested at the larval stage and on. We show that ether interferes with protein integrity in the egg, leading to altered deployment of Hsp90 and widespread repression of Trithorax-mediated establishment of active H3K4me3 chromatin marks throughout the genome. Despite this global reduction, Ubx targets and wing development genes preferentially retain higher levels of H3K4me3 that predispose these genes for later up-regulation in the larval haltere disc, hence the wing-like outcome. Consistent with compromised protein integrity during the exposure, the penetrance of bithorax transformations increases by genetic or chemical reduction of Hsp90 function. Moreover, joint reduction in Hsp90 and trx gene dosage can cause bithorax transformations without exposure to ether, supporting an underlying epistasis between Hsp90 and trx loss-of-functions. These findings implicate environmental disruption of protein integrity at the onset of histone methylation with altered epigenetic regulation of developmental patterning genes. The emerging picture provides a unique example wherein the alleviation of the Hsp90 "capacitor function" by the environment drives a morphogenetic shift towards an ancestral-like body plan. The morphogenetic impact of chaperone response during a major setup of epigenetic patterns may be a general scheme for organ transformation by environmental cues.

Automated Design of Efficient and Functionally Diverse Enzyme Repertoires.

Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput experimental screens and opens the way to designing highly efficient and diverse catalytic repertoires.

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin ß-ODAP in grass pea.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

Carbohydrate dynamics in Populus trees under drought: An expression atlas of genes related to sensing, translocation, and metabolism across organs.

In trees, nonstructural carbohydrates (NSCs) serve as long-term carbon storage and long-distance carbon transport from source to sink. NSC management in response to drought stress is key to our understanding of drought acclimation. However, the molecular mechanisms underlying these processes remain unclear. By combining a transcriptomic approach with NSC quantification in the leaves, stems, and roots of Populus alba under drought stress, we analyzed genes from 29 gene families related to NSC signaling, translocation, and metabolism. We found starch depletion across organs and accumulation of soluble sugars (SS) in the leaves. Activation of the trehalose-6-phosphate/SNF1-related protein kinase (SnRK1) signaling pathway across organs via the suppression of class I TREHALOSE-PHOSPHATE SYNTHASE (TPS) and the expression of class II TPS genes suggested an active response to drought. The expression of SnRK1α and ß subunits, and SUCROSE SYNTHASE6 supported SS accumulation in leaves. The upregulation of active transporters and the downregulation of most passive transporters implied a shift toward active sugar transport and enhanced regulation over partitioning. SS accumulation in vacuoles supports osmoregulation in leaves. The increased expression of sucrose synthesis genes and reduced expression of sucrose degradation genes in the roots did not coincide with sucrose levels, implying local sucrose production for energy. Moreover, the downregulation of invertases in the roots suggests limited sucrose allocation from the aboveground organs. This study provides an expression atlas of NSC-related genes that respond to drought in poplar trees, and can be tested in tree improvement programs for adaptation to drought conditions.

Design and in vitro realization of carbon-conserving photorespiration.

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO2 Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO2 Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO2 release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic-stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO2, even if most of their enzymes operate at a tenth of Rubisco's maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD+, thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbon-conserving photorespiration pathway.

Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro.

The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis.

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.

Efficacy of the rePON1 mutant IIG1 to prevent cyclosarin toxicity in vivo and to detoxify structurally different nerve agents in vitro.

The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of IIG1 (1 mg/kg) prevented systemic toxicity in cyclosarin (~2LD50)-poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity, and protected erythrocyte AChE activity partially. A lower IIG1 dose (0.2 mg/kg) already prevented mortality and reduced systemic toxicity. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent. The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents.

Directed evolution of hydrolases for prevention of G-type nerve agent intoxication.

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ∼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ∼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.

Stable Mammalian Serum Albumins Designed for Bacterial Expression.

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties.

Potential role of phenotypic mutations in the evolution of protein expression and stability.

Phenotypic mutations (errors occurring during protein synthesis) are orders of magnitude more frequent than genetic mutations. Consequently, the sequences of individual protein molecules transcribed and translated from the same gene can differ. To test the effects of such mutations, we established a bacterial system in which an antibiotic resistance gene (TEM-1 beta-lactamase) was transcribed by either a high-fidelity RNA polymerase or its error-prone mutant. This setup enabled the analysis of individual mRNA transcripts that were synthesized under normal or error-prone conditions. We found that an increase of approximately 20-fold in the frequency of transcription errors promoted the evolution of higher TEM-1 expression levels and of more stable enzyme variants. The stabilized variants exhibited a distinct advantage under error-prone transcription, although under normal transcription they conferred resistance similar to wild-type TEM-1. They did so, primarily, by increasing TEM-1's tolerance to destabilizing deleterious mutations that arise from transcriptional errors. The stabilized TEM-1 variants also showed increased tolerance to genetic mutations. Thus, although phenotypic mutations are not individually subjected to inheritance and natural selection, as are genetic mutations, they collectively exert a direct and immediate effect on protein fitness. They may therefore play a role in shaping protein traits such as expression levels, stability, and tolerance to genetic mutations.

The identification and characterization of an oxalyl-CoA synthetase from grass pea (Lathyrus sativus L.).

Oxalic acid is a small metabolite found in many plants. It serves as protection from herbivores, a chelator of metal ions, a regulator of calcium levels, and additional tasks. However, it is also a strong di-carboxylic acid that can compromise plant viability by reducing cellular pH. Several metabolic pathways have evolved to control oxalate levels in plants by enzymatic degradation. Among them is the pathway that utilizes oxalyl-CoA synthetase (OCS, EC 6.2.1.8) and ATP to convert oxalate to oxalyl-CoA. Oxalyl-CoA can then be degraded to CO2 or utilized as a precursor for the synthesis of other compounds. In grass pea (Lathyrus sativus L.), a grain legume grown in Asia and Africa for human and animal consumption, the neurotoxic compound ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP) is synthesized from oxalyl-CoA and l-α,ß-diaminopropionic acid (l-DAPA). Here, we report on the identification and characterization of oxalyl CoA-synthetase from grass pea (LsOCS). The gene encoding LsOCS was amplified from grass pea, and then expressed and purified from E. coli cells as an untagged, monomeric protein of 56 kDa. Its catalytic efficiency with oxalate, K oxalate M = 71.5 ± 13.3 µM, V max = 8.2 ± 0.8 µmole min-1 mg-1, was similar to that of OCS homologs from Arabidopsis thaliana (AtAAE3) and Medicago truncatula (MtAAE3). The enzyme was crystalized in complex with AMP and is the first OCS whose structure was determined in the thioester-forming conformation. Finally, we propose that substituting LsOCS with an oxalate oxidase or decarboxylase may reduce the levels of ß-ODAP in grass pea.

Designed High-Redox Potential Laccases Exhibit High Functional Diversity.

White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for the chemical industry due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative-directed evolution protocols have been applied to improve their expression, activity, and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express. First, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib-designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3-4 subtle mutations that change the polarity, solvation, and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles. These stable and versatile HRPLs are a step toward generating an effective lignin-degrading consortium of enzymes that can be secreted from yeast. The stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.

Designer Liposomic Nanocarriers Are Effective Biofilm Eradicators.

Drug delivery via nanovehicles is successfully employed in several clinical settings, yet bacterial infections, forming microbial communities in the form of biofilms, present a strong challenge to therapeutic treatment due to resistance to conventional antimicrobial therapies. Liposomes can provide a versatile drug-vector strategy for biofilm treatment, but are limited by the need to balance colloidal stability with biofilm penetration. We have discovered a liposomic functionalization strategy, using membrane-embedded moieties of poly[2-(methacryloyloxy)ethyl phosphorylcholine], pMPC, that overcomes this limitation. Such pMPCylation results in liposomic stability equivalent to current functionalization strategies (mostly PEGylation, the present gold-standard), but with strikingly improved cellular uptake and cargo conveyance. Fluorimetry, cryo-electron, and fluorescence microscopies reveal a far-enhanced antibiotic delivery to model Pseudomonas aeruginosa biofilms by pMPC-liposomes, followed by faster cytosolic cargo release, resulting in significantly greater biofilm eradication than either PEGylation or free drug. Moreover, this combination of techniques uncovers the molecular mechanism underlying the enhanced interaction with bacteria, indicating it arises from bridging by divalent ions of the zwitterionic groups on the pMPC moieties to the negatively charged lipopolysaccharide chains emanating from the bacterial membranes. Our results point to pMPCylation as a transformative strategy for liposomal functionalization, leading to next-generation delivery systems for biofilm treatment.

Catalytic bioscavengers as countermeasures against organophosphate nerve agents.

Recent years have seen an increasing number of incidence, in which organophosphate nerve agents (OPNAs) have been used against civilians with devastating outcomes. Current medical countermeasures against OPNA intoxications are aimed at mitigating their symptoms, but are unable to effectively prevent them. In addition, they may fail to prevent the onset of a cholinergic crisis in the brain and its secondary toxic manifestations. The need for improved medical countermeasures has led to the development of bioscavengers; proteins and enzymes that may prevent intoxication by binding and inactivating OPNAs before they can reach their target organs. Non-catalytic bioscavengers such as butyrylcholinesterase, can rapidly bind OPNA molecules in a stoichiometric and irreversible manner, but require the administration of large protein doses to prevent intoxication. Thus, many efforts have been made to develop catalytic bioscavengers that could rapidly detoxify OPNAs without being inactivated in the process. Such enzymes may provide effective prophylactic protection and improve post-exposure treatments using much lower protein doses. Here we review attempts to develop catalytic bioscavengers using molecular biology, directed evolution and enzyme engineering techniques; and natural or computationally designed enzymes. These include both stoichiometric scavengers and enzymes that can hydrolyze OPNAs with low catalytic efficiencies. We discuss the catalytic parameters of evolved and engineered enzymes and the results of in-vivo protection and post-exposure experiments performed using OPNAs and bioscavengers. Finally, we briefly address some of the challenges that need to be met in order to transition these enzymes into clinically approved drugs.

Avoiding and controlling double transformation artifacts.

This article describes a set of standard control experiments for the authentication of new protein variants isolated through library selection and site-directed mutagenesis. These controls are specifically designed to rule out artifacts derived from 'double transformants' -- i.e. cells transformed with, or infected by, two different plasmids simultaneously. These seem to have been the source of past artifacts and, as demonstrated here, are far more common than generally recognized. By following standard protocols for cloning, plasmid isolation, subcloning, in combination with functional assays, the presence of such artifacts can be ruled out. This protocol needs to be applied for any new variant isolated from heterogeneous gene repertoires, and in particular for variants isolated by selection for either enzymatic activity, or binding.

Enzyme engineering: reaching the maximal catalytic efficiency peak.

The practical need for highly efficient enzymes presents new challenges in enzyme engineering, in particular, the need to improve catalytic turnover (kcat) or efficiency (kcat/KM) by several orders of magnitude. However, optimizing catalysis demands navigation through complex and rugged fitness landscapes, with optimization trajectories often leading to strong diminishing returns and dead-ends. When no further improvements are observed in library screens or selections, it remains unclear whether the maximal catalytic efficiency of the enzyme (the catalytic 'fitness peak') has been reached; or perhaps, an alternative combination of mutations exists that could yield additional improvements. Here, we discuss fundamental aspects of the process of catalytic optimization, and offer practical solutions with respect to overcoming optimization plateaus.

Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers.

Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (kcat/KM) of 5 × 106 M-1 min-1. However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of >107 M-1 min-1. Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a kcat/KM ≥ 5 × 107 M-1 min-1 and Russian VX (RVX) with a kcat/KM ≥ 107 M-1 min-1. These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by >5000-fold with VX and by >17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at kcat/KM > 5 × 107 M-1 min-1) and can thus serve as candidates for broad-spectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.