The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond forming reactions should be broadly useful in synthetic chemistry.
The Rosetta software suite for macromolecular modeling, docking, and design is widely used in pharmaceutical, industrial, academic, non-profit, and government laboratories. Despite its broad modeling capabilities, Rosetta remains consistently among leading software suites when compared to other methods created for highly specialized protein modeling and design tasks. Developed for over two decades by a global community of over 60 laboratories, Rosetta has undergone multiple refactorings, and now comprises over three million lines of code. Here we discuss methods developed in the last five years in Rosetta, involving the latest protocols for structure prediction; protein-protein and protein-small molecule docking; protein structure and interface design; loop modeling; the incorporation of various types of experimental data; modeling of peptides, antibodies and proteins in the immune system, nucleic acids, non-standard chemistries, carbohydrates, and membrane proteins. We briefly discuss improvements to the energy function, user interfaces, and usability of the software. Rosetta is available at www.rosettacommons.org.
We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.computational protein design | pathway engineering | carbon fixation N ovel strategies are needed to address current challenges in energy storage and carbon sequestration. One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals. Many synthetic pathways to produce value-added chemicals from common feedstocks, such as glucose, have been constructed in organisms that lack one-carbon anabolic pathways, such as Escherichia coli or Saccharomyces cerevisiae (1-3); however, despite considerable effort, it has been difficult to introduce heterologous one-carbon fixing pathways into these organisms (4). Likely problems include the inherent complexity, environmental sensitivity, inefficiency, or unfavorable chemical driving force of naturally occurring one-carbon metabolic pathways (5).An optimal pathway for one-carbon utilization in common synthetic biology platforms would be (i) composed of a minimal number of enzymes, (ii) linear and disconnected from other metabolic pathways, (iii) thermodynamically favorable with a significant driving force at most or all steps, and (iv) capable of functioning in a robust manner under both aerobic and anaerobic conditions (5). A pathway with these properties could enable the assimilation of one-carbon molecules as the sole carbon source for the production of fuels and chemicals. Although no such pathway is known in nature, the established electrochemical reduction of carbon dioxide to formate under ambient temperatures and pressures in neutral aqueous solutions provides an attractive starting point for a onecarbon fixation pathway (5-8).We describe the computational design of an enzyme that catalyzes the carboligation of three one-carbon molecules into a single three-carbon molecule. This enzyme enables the construction of a new pathway, the formolase pathway, in which formate is converted into the central metabolite dihydroxyacetone phosphate (DHAP; Fig. 1). The use of computational protein design to reengineer catalytic activities opens up the pathway design space beyond that available based o...
Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP + ). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN + ). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 10 7 -fold cofactor specificity switch toward NMN + over NADP + based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the Reprints and permissions information is available at www.nature.com/reprints.
By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed DielsAlderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.biocatalysis | computational enzyme design | Diels-Alder reaction | laboratory evolution | enzyme mechanism
The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a kcat/KM of 568 M–1 s–1, representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.
Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions within physiologically relevant time frames, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease.
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