A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency.

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.

Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor.

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.

Computationally Supported Inversion of Ketoreductase Stereoselectivity.

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6-8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of kcat /KM values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

Substrate Induced Movement of the Metal Cofactor between Active and Resting State.

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M2+ R ), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M2+ A ) at 2.4 Šdistance. Bidentate coordination of a ketoacid substrate to M2+ A affords the overall lowest energy complex, which drives the transition from M2+ R to M2+ A . While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αß)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis.

Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.

The mycobacterial PQS dioxygenase AqdC, a cofactor-less protein with an α/ß-hydrolase fold, inactivates the virulence-associated quorum-sensing signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti-virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in Tappm of >2 °C, they all impaired catalytic activity. Combining substitutions, the proteins AqdC-G40K-A134L-G220D-Y238W and AqdC-G40K-G220D-Y238W showed extended half-lives and the best trade-off between stability and activity, with increases in Tappm of 11.8 and 6.1 °C and relative activities of 22 and 72 %, respectively, compared to AqdC. Molecular dynamics simulations and principal component analysis suggested that stabilized proteins are less flexible than AqdC, and the loss of catalytic activity likely correlates with an inability to effectively open the entrance to the active site.

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.

Computational Prediction of ω-Transaminase Specificity by a Combination of Docking and Molecular Dynamics Simulations.

ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.

Computational Design of Enantiocomplementary Epoxide Hydrolases for Asymmetric Synthesis of Aliphatic and Aromatic Diols.

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis-stilbene oxide, and enantiocomplementary mutants produced (S,S)- and (R,R)-stilbene diol with >97 % enantiomeric excess. An (R,R)-selective mutant was used to prepare (R,R)-stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee). Some variants displayed higher catalytic rates (kcat ) than the original enzyme, but in most cases KM values increased as well. The results demonstrate the feasibility of computational design and screening to engineer enantioselective epoxide hydrolase variants with very limited laboratory screening.

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.

We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C1 ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH- ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications.

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor.

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.

Computational redesign of enzymes for regio- and enantioselective hydroamination.

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 ß-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%, ß-regioselectivity >99% and product enantiomeric excess >99%. The results highlight the use of computational design to rapidly adapt an enzyme to industrially viable reactions.

Assessment of the interaction between the flux-signaling metabolite fructose-1,6-bisphosphate and the bacterial transcription factors CggR and Cra.

Bacteria regulate cell physiology in response to extra- and intracellular cues. Recent work showed that metabolic fluxes are reported by specific metabolites, whose concentrations correlate with flux through the respective metabolic pathway. An example of a flux-signaling metabolite is fructose-1,6-bisphosphate (FBP). In turn, FBP was proposed to allosterically regulate master regulators of carbon metabolism, Cra in Escherichia coli and CggR in Bacillus subtilis. However, a number of questions on the FBP-mediated regulation of these transcription factors is still open. Here, using thermal shift assays and microscale thermophoresis we demonstrate that FBP does not bind Cra, even at millimolar physiological concentration, and with electrophoretic mobility shift assays we also did not find FBP-mediated impairment of Cra's affinity for its operator site, while fructose-1-phosphate does. Furthermore, we show for the first time that FBP binds CggR within the millimolar physiological concentration range of the metabolite, and decreases DNA-binding activity of this transcription factor. Molecular docking experiments only identified a single FBP binding site CggR. Our results provide the long thought after clarity with regards to regulation of Cra activity in E. coli and reveals that E. coli and B. subtilis use distinct cellular mechanism to transduce glycolytic flux signals into transcriptional regulation.

Efficient Enzymatic Cyclization of Disulfide-Rich Peptides by Using Peptide Ligases.

Disulfide-rich macrocyclic peptides-cyclotides, for example-represent a promising class of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale still represents a major challenge. Here we report new chemoenzymatic strategies using peptide ligase variants-inter alia, omniligase-1-for the efficient and scalable one-pot cyclization and folding of the native cyclotides MCoTI-II, kalata B1 and variants thereof, as well as of the θ-defensin RTD-1. The synthesis of the kB1 variant T20K was successfully demonstrated at multi-gram scale. The existence of several ligation sites for each macrocycle makes this approach highly flexible and facilitates both the larger-scale manufacture and the engineering of bioactive, grafted cyclotide variants, therefore clearly offering a valuable and powerful extension of the existing toolbox of enzymes for peptide head-to-tail cyclization.

Design of a substrate-tailored peptiligase variant for the efficient synthesis of thymosin-α1.

The synthesis of thymosin-α1, an acetylated 28 amino acid long therapeutic peptide, via conventional chemical methods is exceptionally challenging. The enzymatic coupling of unprotected peptide segments in water offers great potential for a more efficient synthesis of peptides that are difficult to synthesize. Based on the design of a highly engineered peptide ligase, we developed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the production of thymosin-α1via a 14-mer + 14-mer segment condensation strategy. Using structure-inspired enzyme engineering, the thiol-subtilisin variant peptiligase was tailored to recognize the respective 14-mer thymosin-α1 segments in order to create a clearly improved biocatalyst, termed thymoligase. Thymoligase catalyzes peptide bond formation between both segments with a very high efficiency (>94% yield) and is expected to be well applicable to many other ligations in which residues with similar characteristics (e.g. Arg and Glu) are present in the respective positions P1 and P1'. The crystal structure of thymoligase was determined and shown to be in good agreement with the model used for the engineering studies. The combination of the solid phase peptide synthesis (SPPS) of the 14-mer segments and their thymoligase-catalyzed ligation on a gram scale resulted in a significantly increased, two-fold higher overall yield (55%) of thymosin-α1 compared to those typical of existing industrial processes.

Real-Time Conformational Changes and Controlled Orientation of Native Proteins Inside a Protein Nanoreactor.

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.

X-ray crystallographic validation of structure predictions used in computational design for protein stabilization.

Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side-chain conformations were correctly predicted for 72-88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c-Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B-FIT methods.

Metabolism of ß-valine via a CoA-dependent ammonia lyase pathway.

Pseudomonas species strain SBV1 can rapidly grow on medium containing ß-valine as a sole nitrogen source. The tertiary amine feature of ß-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of ß-valine, a PsSBV1 gene library was prepared and used to complement the ß-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding ß-valinyl-coenzyme A ligase (BvaA) and two genes encoding ß-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate ß-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only ß-valine and to a lesser extent, 3-aminobutyrate and ß-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate ß-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

Enantioselective enzymes by computational design and in silico 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 library design for increasing haloalkane dehalogenase stability.

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app , 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.