Enzymatic Conversion of CO2: From Natural to Artificial Utilization.

Enzymatic carbon dioxide fixation is one of the most important metabolic reactions as it allows the capture of inorganic carbon from the atmosphere and its conversion into organic biomass. However, due to the often unfavorable thermodynamics and the difficulties associated with the utilization of CO2, a gaseous substrate that is found in comparatively low concentrations in the atmosphere, such reactions remain challenging for biotechnological applications. Nature has tackled these problems by evolution of dedicated CO2-fixing enzymes, i.e., carboxylases, and embedding them in complex metabolic pathways. Biotechnology employs such carboxylating and decarboxylating enzymes for the carboxylation of aromatic and aliphatic substrates either by embedding them into more complex reaction cascades or by shifting the reaction equilibrium via reaction engineering. This review aims to provide an overview of natural CO2-fixing enzymes and their mechanistic similarities. We also discuss biocatalytic applications of carboxylases and decarboxylases for the synthesis of valuable products and provide a separate summary of strategies to improve the efficiency of such processes. We briefly summarize natural CO2 fixation pathways, provide a roadmap for the design and implementation of artificial carbon fixation pathways, and highlight examples of biocatalytic cascades involving carboxylases. Additionally, we suggest that biochemical utilization of reduced CO2 derivates, such as formate or methanol, represents a suitable alternative to direct use of CO2 and provide several examples. Our discussion closes with a techno-economic perspective on enzymatic CO2 fixation and its potential to reduce CO2 emissions.

Expanding the structural analysis capabilities on an Orbitrap-based mass spectrometer for large macromolecular complexes.

Native mass spectrometry can provide insight into the structure of macromolecular biological systems. As analytes under investigation get larger and more complex, instrument capabilities need to be advanced. Herein, modifications to an Orbitrap Q Exactive Plus mass spectrometer that increase signal intensity, mass resolution, and maximum m/z measurable are described.

Optimization of Enzyme Mechanism along the Evolutionary Trajectory of a Computationally Designed (Retro-)Aldolase.

De novo biocatalysts have been successfully generated by computational design and subsequent experimental optimization. Here, we examined the evolutionary history of the computationally designed (retro-)aldolase RA95. The modest activity of the starting enzyme was previously improved 105-fold over many rounds of mutagenesis and screening to afford a proficient biocatalyst for enantioselective cleavage and synthesis of ß-hydroxyketones. Using a set of representative RA95 variants, we probed individual steps in the multistep reaction pathway to determine which processes limit steady-state turnover and how mutations that accumulated along the evolutionary trajectory influenced the kinetic mechanism. We found that the overall rate-limiting step for aldol cleavage shifted from C-C bond scission (or an earlier step in the pathway) for the computational design to product release for the evolved enzymes. Specifically, interconversion of Schiff base and enamine intermediates, formed covalently between acetone and the catalytic lysine residue, was found to be the slowest step for the most active variants. A complex hydrogen bond network of four active site residues, which was installed in the late stages of laboratory evolution, apparently enhances lysine reactivity and facilitates efficient proton shuffling. This catalytic tetrad accounts for the tremendous rate acceleration observed for all steps of the mechanism, most notably Schiff base formation and hydrolysis. Comparison of our results with kinetic and structural studies on natural aldolases provides valuable feedback for computational enzyme design and laboratory evolution approaches alike.

The C-terminal peptide of Aquifex aeolicus riboflavin synthase directs encapsulation of native and foreign guests by a cage-forming lumazine synthase.

Encapsulation of specific enzymes in self-assembling protein cages is a hallmark of bacterial compartments that function as counterparts to eukaryotic organelles. The cage-forming enzyme lumazine synthase (LS) from Bacillus subtilis (BsLS), for example, encapsulates riboflavin synthase (BsRS), enabling channeling of lumazine from the site of its generation to the site of its conversion to vitamin B2 Elucidating the molecular mechanisms underlying the assembly of these supramolecular complexes could help inform new approaches for metabolic engineering, nanotechnology, and drug delivery. To that end, we investigated a thermostable LS from Aquifex aeolicus (AaLS) and found that it also forms cage complexes with the cognate riboflavin synthase (AaRS) when both proteins are co-produced in the cytosol of Escherichia coli A 12-amino acid-long peptide at the C terminus of AaRS serves as a specific localization sequence responsible for targeting the guest to the protein compartment. Sequence comparisons suggested that analogous peptide segments likely direct RS complexation by LS cages in other bacterial species. Covalent fusion of this peptide tag to heterologous guest molecules led to their internalization into AaLS assemblies both in vivo and in vitro, providing a firm foundation for creating tailored biomimetic nanocompartments for medical and biotechnological applications.

Structure and assembly of scalable porous protein cages.

Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of two versatile encapsulation systems that exploit engineered electrostatic interactions for cargo loading. We show that increasing the number of negative charges on the lumenal surface of lumazine synthase, a protein that naturally assembles into a ∼1-MDa dodecahedron composed of 12 pentamers, induces stepwise expansion of the native protein shell, giving rise to thermostable ∼3-MDa and ∼6-MDa assemblies containing 180 and 360 subunits, respectively. Remarkably, these expanded particles assume unprecedented tetrahedrally and icosahedrally symmetric structures constructed entirely from pentameric units. Large keyhole-shaped pores in the shell, not present in the wild-type capsid, enable diffusion-limited encapsulation of complementarily charged guests. The structures of these supercharged assemblies demonstrate how programmed electrostatic effects can be effectively harnessed to tailor the architecture and properties of protein cages.

Irreversible Cysteine-Selective Protein Labeling Employing Modular Electrophilic Tetrafluoroethylation Reagents.

Fluoroalkylation reagents based on hypervalent iodine are widely used to transfer fluoroalkyl moieties to various nucleophiles. However, the transferred groups have so far been limited to simple structural motifs. We herein report a reagent featuring a secondary amine that can be converted to amide, sulfonamide, and tertiary amine derivatives in one step. The resulting reagents bear manifold functional groups, many of which would not be compatible with the original synthetic pathway. Exploiting this structural versatility and the known high reactivity toward thiols, the new-generation reagents were used in bioconjugation with an artificial retro-aldolase, containing an exposed cysteine and a reactive catalytic lysine. Whereas commercial reagents based on maleimide and iodoacetamide labeled both sites, the iodanes exclusively modified the cysteine residue. The study thus demonstrates that modular fluoroalkylation reagents can be used as tools for cysteine-selective bioconjugation.

Quantitative Packaging of Active Enzymes into a Protein Cage.

Genetic fusion of cargo proteins to a positively supercharged variant of green fluorescent protein enables their quantitative encapsulation by engineered lumazine synthase capsids possessing a negatively charged lumenal surface. This simple tagging system provides a robust and versatile means of creating hierarchically ordered protein assemblies for use as nanoreactors. The generality of the encapsulation strategy and its effect on enzyme function were investigated with eight structurally and mechanistically distinct catalysts.

Diffusion-Limited Cargo Loading of an Engineered Protein Container.

The engineered bacterial nanocompartment AaLS-13 is a promising artificial encapsulation system that exploits electrostatic interactions for cargo loading. In order to study its ability to take up and retain guests, a pair of fluorescent proteins was developed which allows spectroscopic determination of the extent of encapsulation by Förster resonance energy transfer (FRET). The encapsulation process is generally complete within a second, suggesting low energetic barriers for proteins to cross the capsid shell. Formation of intermediate aggregates upon mixing host and guest in vitro complicates capsid loading at low ionic strength, but can be sidestepped by increasing salt concentrations or diluting the components. Encapsulation of guests is completely reversible, and the position of the equilibrium is easily tuned by varying the ionic strength. These results, which challenge the notion that AaLS-13 is a continuous rigid shell, provide valuable information about cargo loading that will guide ongoing efforts to engineer functional host-guest complexes. Moreover, it should be possible to adapt the protein FRET pair described in this report to characterize functional capsid-cargo complexes generated by other encapsulation systems.