•Targeting critical sites for stability improvement.•Consensus/ancestral sequence based protein stabilization.•Computational design of thermostability.•Loop grafting and chimeras for stabilization.
...Protein engineering strategies for increasing stability can be improved by replacing random mutagenesis and high-throughput screening by approaches that include bioinformatics and computational design. Mutations can be focused on regions in the structure that are most flexible and involved in the early steps of thermal unfolding. Sequence analysis can often predict the position and nature of stabilizing mutations, and may allow the reconstruction of thermostable ancestral sequences. Various computational tools make it possible to design stabilizing features, such as hydrophobic clusters and surface charges. Different methods for designing chimeric enzymes can also support the engineering of more stable proteins without the need of high-throughput screening.
Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase ...variants for the production of highly enantioenriched (S,S)‐diols and (R,R)‐diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high‐throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.
Computational enzyme design holds great promise for providing tailored biocatalysts. A strategy is presented that makes it possible to obtain enantiodivergent and highly enantioselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.
Protein conformations play crucial roles in most, if not all, biological processes. Here we show that the current carried through a nanopore by ions allows monitoring conformational changes of single ...and native substrate-binding domains (SBD) of an ATP-Binding Cassette importer in real-time. Comparison with single-molecule Förster Resonance Energy Transfer and ensemble measurements revealed that proteins trapped inside the nanopore have bulk-like properties. Two ligand-free and two ligand-bound conformations of SBD proteins were inferred and their kinetic constants were determined. Remarkably, internalized proteins aligned with the applied voltage bias, and their orientation could be controlled by the addition of a single charge to the protein surface. Nanopores can thus be used to immobilize proteins on a surface with a specific orientation, and will be employed as nanoreactors for single-molecule studies of native proteins. Moreover, nanopores with internal protein adaptors might find further practical applications in multianalyte sensing devices.
The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse ...stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10–12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85°C, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution.
Introduction of innovative biocatalytic processes offers great promise for applications in green chemistry. However, owing to limited catalytic performance, the enzymes harvested from nature's ...biodiversity often need to be improved for their desired functions by time-consuming iterative rounds of laboratory evolution. Here we describe the use of structure-based computational enzyme design to convert Bacillus sp. YM55-1 aspartase, an enzyme with a very narrow substrate scope, to a set of complementary hydroamination biocatalysts. The redesigned enzymes catalyze asymmetric addition of ammonia to substituted acrylates, affording enantiopure aliphatic, polar and aromatic β-amino acids that are valuable building blocks for the synthesis of pharmaceuticals and bioactive compounds. Without a requirement for further optimization by laboratory evolution, the redesigned enzymes exhibit substrate tolerance up to a concentration of 300 g/L, conversion up to 99%, β-regioselectivity >99% and product enantiomeric excess >99%. The results highlight the use of computational design to rapidly adapt an enzyme to industrially viable reactions.
Nitroreductases (NRs) are NAD(P)H‐dependent flavoenzymes that reduce nitro aromatic compounds to their corresponding arylamines via the nitroso and hydroxylamine intermediates. Because of their broad ...substrate scope and versatility, NRs have found application in multiple fields such as biocatalysis, bioremediation, cell‐imaging and prodrug activation. However, only a limited number of members of the broad NR superfamily (> 24 000 sequences) have been experimentally characterized. Within this group of enzymes, only few are capable of amine synthesis, which is a fundamental chemical transformation for the pharmaceutical, agricultural, and textile industries. Herein, we provide a comprehensive description of a recently discovered NR from Bacillus tequilensis , named BtNR. This enzyme has previously been demonstrated to have the capability to fully convert nitro aromatic and heterocyclic compounds to their respective primary amines. In this study, we determined its biochemical, kinetic and structural properties, including its apparent melting temperature ( T m ) of 59 °C, broad pH activity range (from pH 3 to 10) and a notably low redox potential (−236 ± 1 mV) in comparison to other well‐known NRs. We also determined its steady‐state and pre‐steady‐state kinetic parameters, which are consistent with other NRs. Additionally, we elucidated the crystal structure of BtNR, which resembles the well‐characterized Escherichia coli oxygen‐insensitive NAD(P)H nitroreductase (NfsB), and investigated the substrate binding in its active site through docking and molecular dynamics studies with four nitro aromatic substrates. Guided by these structural analyses, we probed the functional roles of active site residues by site‐directed mutagenesis. Our findings provide valuable insights into the biochemical and structural properties of BtNR, as well as its potential applications in biotechnology.
Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its ...emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single‐cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell‐intrinsic correlation between glycolytic flux and levels of fructose‐1,6‐bisphosphate (FBP), we transplanted the B. subtilis FBP‐binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.
Synopsis
The study presents a biosensor of glycolytic flux in single yeast cells, based on detecting fructose‐1,6‐phosphate (FBP) as a flux‐signalling metabolite, using the bacterial FBP‐responsive transcription factor CggR. FBP levels and glycolytic flux can be measured with flow cytometry and microscopy.
A synthetic yeast promoter regulated by the FBP‐responsive bacterial transcription factor CggR was developed.
The FBP binding site of the transcription factor was engineered to increase the sensor's dynamic range.
Growth‐independent expression levels of the transcription factor were established.
The sensor can be used to reveal differences in FBP levels and glycolytic flux.
The study presents a biosensor of glycolytic flux in single yeast cells, based on detecting fructose‐1,6‐phosphate (FBP) as a flux‐signalling metabolite, using the bacterial FBP‐responsive transcription factor CggR. FBP levels and glycolytic flux can be measured with flow cytometry and microscopy.
Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin ...transferase and carving a flavinylation motif in target proteins, we demonstrate that “dissociable” flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.
Computational approaches could decrease the need for the laborious high-throughput experimental screening that is often required to improve enzymes by mutagenesis. Here, we report that using multiple ...short molecular dynamics (MD) simulations makes it possible to accurately model enantioselectivity for large numbers of enzyme–substrate combinations at low computational costs. We chose four different haloalkane dehalogenases as model systems because of the availability of a large set of experimental data on the enantioselective conversion of 45 different substrates. To model the enantioselectivity, we quantified the frequency of occurrence of catalytically productive conformations (near attack conformations) for pairs of enantiomers during MD simulations. We found that the angle of nucleophilic attack that leads to carbon–halogen bond cleavage was a critical variable that limited the occurrence of productive conformations; enantiomers for which this angle reached values close to 180° were preferentially converted. A cluster of 20–40 very short (10 ps) MD simulations allowed adequate conformational sampling and resulted in much better agreement to experimental enantioselectivities than single long MD simulations (22 ns), while the computational costs were 50–100 fold lower. With single long MD simulations, the dynamics of enzyme–substrate complexes remained confined to a conformational subspace that rarely changed significantly, whereas with multiple short MD simulations a larger diversity of conformations of enzyme–substrate complexes was observed.
Enzymes are crucial for the emergence and sustenance of life on earth. How they became catalytically active during their evolution is still an open question. Two opposite explanations are plausible: ...acquiring a mechanism in a series of discrete steps or all at once in a single evolutionary event. Here, we use molecular phylogeny, ancestral sequence reconstruction, and biochemical characterization to follow the evolution of a specialized group of flavoprotein monooxygenases, the bacterial Baeyer-Villiger monooxygenases (BVMOs). These enzymes catalyze an intricate chemical reaction relying on three different elements: a reduced nicotinamide cofactor, dioxygen, and a substrate. Characterization of ancestral BVMOs shows that the catalytic mechanism evolved in a series of steps starting from a FAD-binding protein and further acquiring reactivity and specificity toward each of the elements participating in the reaction. Together, the results of our work portray how an intrinsically complex catalytic mechanism emerged during evolution.
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•BVMOs’ mechanism evolved through a series of slightly overlapped, discrete steps•Catalytic elements of the mechanism were recruited and later function optimized•The hydride transfer process precedes establishment of the nicotinamide specificity•AncBVMOs evolved oxygen reactivity contingent on the hydride donor specificity
Yang et al. report how the complex catalytic mechanism of bacterial BVMOs evolved. AncBVMOs increased their activity over time contingent on the hydride donor usage and improvement of oxygen reactivity. The network of residues in the active site, the NADP+ cofactor, and global conformational changes govern the functionality.
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