Site-Specific Conjugation of Bottlebrush Polymers to Therapeutic Protein via Bioorthogonal Chemistry.

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.

Multivalent Albumin-Neonatal Fc Receptor Interactions Mediate a Prominent Extension of the Serum Half-Life of a Therapeutic Protein.

Human serum albumin (HSA) has been used to extend the serum half-life of therapeutic proteins owing to its exceptionally long serum half-life via the neonatal Fc receptor (FcRn)-mediated recycling mechanism. In most cases, only one HSA molecule was conjugated to a therapeutic protein, leading to a limited extension of the serum half-life. In this study, we hypothesized that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. We chose urate oxidase (Uox), a tetrameric therapeutic protein used for the treatment of gout, as a model. In previous studies, only one HSA molecule was site-specifically conjugated to one Uox because of poor conjugation yield of the relatively slow bio-orthogonal chemistry, strain-promoted azide-alkyne cycloaddition (SPAAC). To increase the number of HSA molecules conjugated to one Uox, we employed the faster bio-orthogonal chemistry, inverse electron demand Diels-Alder reaction (IEDDA). We site-specifically introduced the phenylalanine analog with a fast-reacting tetrazine group (frTet) into position 174 of each subunit of Uox. We then achieved site-specific HSA conjugation to each subunit of Uox via IEDDA, generating Uox conjugated to four HSA molecules (Uox-HSA4), with a small portion of Uox conjugated to three HSA molecules (Uox-HSA3). We characterized Uox-HSA4 as well as Uox variants conjugated to one or two HSA molecules prepared via SPAAC (Uox-HSA1 or Uox-HSA2). The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. We found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. In particular, the conjugation of four HSA molecules to Uox led to a prominent extension of the serum half-life (over 21 h), which is about 16-fold longer than that of Uox-WT.

Temporal Control of Efficient In Vivo Bioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand Diels-Alder Reaction.

An inverse-electron-demand Diels-Alder (IEDDA) reaction using genetically encoded tetrazine variants enables rapid bioconjugation for diverse applications in vitro and in cellulo. However, in vivo bioconjugation using genetically encoded tetrazine variants is challenging, because the IEDDA coupling reaction competes with rapid elimination of reaction partners in vivo. Here, we tested the hypothesis that a genetically encoded phenylalanine analogue containing a hydrogen-substituted tetrazine (frTet) would increase the IEDDA reaction rate, thereby allowing for successful bioconjugation in vivo. We found that the in vitro IEDDA reaction rate of superfolder green fluorescent protein (sfGFP) containing frTet (sfGFP-frTet) was 12-fold greater than that of sfGFP containing methyl-substituted tetrazine (sfGFP-Tet_v2.0). Additionally, sfGFP variants encapsulated with chitosan-modified, pluronic-based nanocarriers were delivered into nude mice or tumor-bearing mice for in vivo imaging. The in vivo-delivered sfGFP-frTet exhibited almost complete fluorescence recovery upon addition of trans-cyclooctene via the IEDDA reaction within 2 h, whereas sfGFP-Tet_v2.0 did not show substantial fluorescence recovery. These results demonstrated that the genetically encoded frTet allows an almost complete IEDDA reaction in vivo upon addition of trans-cyclooctene, enabling temporal control of in vivo bioconjugation in a very high yield.

Enhanced production of Dopa-incorporated mussel adhesive protein using engineered translational machineries.

Mussel adhesive proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows for production of Dopa-incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannaschii TyrRS (MjTyrRS-Dopa), we altered the anticodon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyrAUA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyrAUA increased the production yield of Dopa-incorporated MAP foot protein type 3 (dfp-3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp-3. Even with coexpression of Dopa-recognizing TyrRSs, dfp-3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids.

The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.

Site-Specific Albumination as an Alternative to PEGylation for the Enhanced Serum Half-Life in Vivo.

Polyethylene glycol (PEG) has been widely used as a serum half-life extender of therapeutic proteins. However, due to immune responses and low degradability of PEG, developing serum half-life extender alternatives to PEG is required. Human serum albumin (HSA) has several beneficial features as a serum half-life extender, including a very long serum half-life, good degradability, and low immune responses. In order to further evaluate the efficacy of HSA, we compared the extent of serum half-life extension of a target protein, superfolder green fluorescent protein (sfGFP), upon HSA conjugation with PEG conjugation side-by-side. Combination of site-specific incorporation of p-azido-l-phenylalanine into sfGFP and copper-free click chemistry achieved the site-specific conjugation of a single HSA, 20 kDa PEG, or 30 kDa PEG to sfGFP. These sfGFP conjugates exhibited the fluorescence comparable to or even greater than that of wild-type sfGFP (sfGFP-WT). In mice, HSA-conjugation to sfGFP extended the serum half-life 9.0 times compared to that of unmodified sfGFP, which is comparable to those of PEG-conjugated sfGFPs (7.3 times for 20 kDa PEG and 9.5 times for 30 kDa PEG). These results clearly demonstrated that HSA was as effective as PEG in extending the serum half-life of a target protein. Therefore, with the additional favorable features, HSA is a good serum half-life extender of a (therapeutic) protein as an alternative to PEG.

Determining binding sites of polycyclic aromatic small molecule-based amyloid-beta peptide aggregation modulators using sequence-specific antibodies.

Numerous aromatic small molecule modulators of amyloid-beta peptide (Aß) monomer aggregation and neurotoxicity have been identified with the ultimate goal of Alzheimer's disease (AD) treatment. Determining binding sites of these modulators on Aß monomer is an important topic in the mechanistic understanding of AD pathology and drug development. However, Aß monomer binding sites have been reported for only a very limited number of Aß modulators. In this article, we present a convenient method for determining aggregation-modulating polycyclic aromatic small molecule ligand binding sites on Aß monomer using immunostaining with a panel of Aß sequence-specific antibodies. To validate our technique, we first examined one modulating aromatic ligand, Congo Red, with known binding sites, which yielded consistent results with previous findings. Then, using the same technique, binding sites on Aß of four known Aß monomer aggregation modulators, Erythrosin B, Eosin Y, Phloxine B, and Rose Bengal, were determined. The identified ligand binding sites were also confirmed by a separate fluorescence quenching-based assay using a panel of overlapping Aß sub-fragments. The technique described here greatly increases researchers' ability to determine the Aß monomer binding site(s) of aggregation-modulating aromatic small molecule ligands and to screen for new ligands that bind specific regions on Aß.

Effects of Non-Natural Amino Acid Incorporation into the Enzyme Core Region on Enzyme Structure and Function.

Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins containing new building blocks with unique structures, chemistry, and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb critical function of a target protein. This study investigates how the site-specific incorporation of NNAAs affects catalytic properties of an enzyme. A NNAA with a hydrophobic and bulky sidechain, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at six different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals volume upon 2Nal incorporation exhibited a greater reduction in the catalytic efficiency. Similarly, the steric incompatibility calculated using RosettaDesign, a protein stability calculation program, correlated with the changes in the catalytic efficiency.

Site-specific bioconjugation of an organometallic electron mediator to an enzyme with retained photocatalytic cofactor regenerating capacity and enzymatic activity.

Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM) has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(P)H being readily available to a redox enzyme, when the local NAD(P)H concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH). A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

Targeting of heparanase-modified syndecan-1 by prosecretory mitogen lacritin requires conserved core GAGAL plus heparan and chondroitin sulfate as a novel hybrid binding site that enhances selectivity.

Cell surface heparan sulfate (HS) proteoglycans shape organogenesis and homeostasis by capture and release of morphogens through mechanisms largely thought to exclude the core protein domain. Nevertheless, heparanase deglycanation of the N-terminal HS-rich domain of syndecan-1 (SDC1), but not SDC2 or -4, is a prerequisite for binding of the prosecretory mitogen lacritin (Ma, P., Beck, S. L., Raab, R. W., McKown, R. L., Coffman, G. L., Utani, A., Chirico, W. J., Rapraeger, A. C., and Laurie, G. W. (2006) Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin. J. Cell Biol. 174, 1097-1106). We now report that the conserved and hydrophobic GAGAL domain in SDC1, adjacent to predicted HS substitution sites, is necessary to ligate and substantially enhance the α-helicity of the amphipathic C terminus of lacritin. Swapping out GAGAL for GADED in SDC2 or for GDLDD in SDC4 (both less hydrophobic) abrogated binding. HS and chondroitin sulfate are also essential. Both are detected in the N terminus, and when incubated with antibodies HS4C3 (anti-HS) or IO3H10 (anti-chondroitin sulfate), binding was absent, as occurred when all three N-terminal glycosaminoglycan substitution sites were mutated to alanine or when cells were treated with 4-methylumbelliferyl-ß-d-xylopyranoside or chlorate to suppress glycosaminoglycan substitution or sulfation, respectively. SDC1 interacts with the hydrophobic face of lacritin via Leu-108/Leu-109/Phe-112 as well as with Glu-103/Lys-107 and Lys-111 of the largely cationic face. Carving a hybrid hydrophobic/electrostatic docking site out of SDC1 in a manner dependent on endogenous heparanase is a dynamic process appropriate for subtle or broad epithelial regulation in morphogenesis, health, and disease.

Cis-suppression to arrest protein aggregation in mammalian cells.

Protein misfolding and aggregation are implicated in numerous human diseases and significantly lower production yield of proteins expressed in mammalian cells. Despite the importance of understanding and suppressing protein aggregation in mammalian cells, a protein design and selection strategy to modulate protein misfolding/aggregation in mammalian cells has not yet been reported. In this work, we address the particular challenge presented by mutation-induced protein aggregation in mammalian cells. We hypothesize that an additional mutation(s) can be introduced in an aggregation-prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation (cis-suppression). As a model protein, we chose human copper, zinc superoxide dismutase mutant (SOD1(A4V) ) containing an alanine to valine mutation at residue 4, associated with the familial form of amyotrophic lateral sclerosis. We used the program RosettaDesign to identify Phe20 in SOD1(A4V) as a key residue responsible for SOD1(A4V) conformational destabilization. This information was used to rationally develop a pool of candidate mutations at the Phe20 site. After two rounds of mammalian-cell based screening of the variants, three novel SOD1(A4V) variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1(A4V) variants were verified using cell fractionation and in vitro stability assays.

Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation.

Mutations introduced to wild-type proteins naturally, or intentionally via protein engineering, often lead to protein aggregation. In particular, protein aggregation within mammalian cells has significant implications in the disease pathology and biologics production; making protein aggregation modulation within mammalian cells a very important engineering topic. Previously, we showed that the semi-rational design approach can be used to reduce the intracellular aggregation of a protein by recovering the conformational stability that was lowered by the mutation. However, this approach has limited utility when no rational design approach to enhance conformational stability is readily available. In order to overcome this limitation, we investigated whether the modification of residues significantly displaced upon the original mutation is an effective way to reduce protein aggregation in mammalian cells. As a model system, human copper, zinc superoxide dismutase mutant containing glycine to alanine mutation at position 93 (SOD1G93A) was used. A panel of mutations was introduced into residues substantially displaced upon the G93A mutation. By using cell-based aggregation assays, we identified several novel variants of SOD1G93A with reduced aggregation propensity within mammalian cells. Our findings successfully demonstrate that the aggregation of a mutant protein can be suppressed by mutating the residues significantly displaced upon the original mutation.

Recovery of rare earth elements from low-grade coal fly ash using a recyclable protein biosorbent.

Rare earth elements (REEs), including those in the lanthanide series, are crucial components essential for clean energy transitions, but they originate from geographically limited regions. Exploiting new and diverse supply sources is vital to facilitating a clean energy future. Hence, we explored the recovery of REEs from coal fly ash (FA), a complex, low-grade industrial feedstock that is currently underutilized (leachate concentrations of REEs in FA are < 0.003 mol%). Herein, we demonstrated the thermo-responsive genetically encoded REE-selective elastin-like polypeptides (RELPs) as a recyclable bioengineered protein adsorbent for the selective retrieval of REEs from coal fly ash over multiple cycles. The results showed that RELPs could be efficiently separated using temperature cycling and reused with high stability, as they retained ∼95% of their initial REE binding capacity even after four cycles. Moreover, RELPs selectively recovered high-purity REEs from the simulated solution containing one representative REE in the range of 0.0001-0.005 mol%, resulting in up to a 100,000-fold increase in REE purity. This study offers a sustainable approach to diversifying REE supplies by recovering REEs from low-grade coal fly ash in industrial wastes and provides a scientific basis for the extraction of high-purity REEs for industrial purposes.

AlbuCatcher for Long-Acting Therapeutics.

Therapeutic proteins, pivotal for treating diverse human diseases due to their biocompatibility and high selectivity, often face challenges such as rapid serum clearance, enzymatic degradation, and immune responses. To address these issues and enable prolonged therapeutic efficacy, techniques to extend the serum half-life of therapeutic proteins are crucial. The AlbuCatcher, a conjugate of human serum albumin (HSA) and SpyCatcher, was proposed as a general technique to extend the serum half-life of diverse therapeutic proteins. HSA, the most abundant blood protein, exhibits a long intrinsic half-life through Fc receptor (FcRn)-mediated recycling. The SpyTag/SpyCatcher (ST/SC) system, known for forming irreversible isopeptide bonds, was employed to conjugate HSA and therapeutic proteins. Site-specific HSA conjugation to SC was achieved using an inverse electron-demand Diels-Alder (IEDDA) reaction, minimizing activity loss. Using urate oxidase (Uox) as a model protein with a short half-life, the small ST was fused to generate Uox-ST. Then, HSA-conjugated Uox (Uox-HSA) was successfully prepared via the Uox-ST/AlbuCatcher reaction. In vitro enzyme assays demonstrated that the impact of ST fusion and HSA conjugation on Uox enzymatic activity is negligible. Pharmacokinetics studies in mice revealed that Uox-HSA exhibits a significantly longer serum half-life (about 18 h) compared to Uox-WT (about 2 h). This extended half-life is attributed to FcRn-mediated recycling of HSA-conjugated Uox, demonstrating the effectiveness of the AlbuCatcher strategy in enhancing the pharmacokinetics of therapeutic proteins.

Enhanced anti-tumor activity of arginine decarboxylase through the incorporation of aromatic amino acids at the multimer-forming interface.

The pressing challenge of cancer's high mortality and invasiveness demands improved therapeutic approaches. Targeting the nutrient dependencies within cancer cells has emerged as a promising approach. This study is dedicated to demonstrating the potential of arginine depletion for cancer treatment. Notably, the focus centers on arginine decarboxylase (RDC), a pH-dependent enzyme expecting enhanced activity within the slightly acidic microenvironments of tumors. To investigate the effect of a single-site mutation on the catalytic efficacy of RDC, diverse amino acids, including glycine, alanine, phenylalanine, tyrosine, tryptophan, p-azido-phenylalanine, and a phenylalanine analog with a hydrogen-substituted tetrazine, were introduced at the crucial threonine site (position 39) in the multimer-forming interface. Remarkably, the introduction of either a natural or a non-natural aromatic amino acid at position 39 substantially boosted enzymatic activity, while amino acids with smaller side chains did not show the same effect. This enhanced enzymatic activity is likely attributed to the reinforced formation of multimer structures through favorable interactions between the introduced aromatic amino acid and the neighboring subunit. Noteworthy, at slightly acidic pH, the RDC variant featuring tryptophan at position 39 demonstrated augmented cytotoxicity against tumor cells compared to the wild-type RDC. This attribute aligns with the tumor microenvironment and positions these variants as potential candidates for targeted cancer therapy.

Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation.

BACKGROUND: The use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties. RESULTS: In this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv. CONCLUSIONS: The antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.

Intracellular Flux Prediction of Recombinant Escherichia coli Producing Gamma-Aminobutyric Acid.

Genome-scale metabolic model (GEM) can be used to simulate cellular metabolic phenotypes under various environmental or genetic conditions. This study utilized the GEM to observe the internal metabolic fluxes of recombinant Escherichia coli producing gamma-aminobutyric acid (GABA). Recombinant E. coli was cultivated in a fermenter under three conditions: pH 7, pH 5, and additional succinic acids. External fluxes were calculated from cultivation results, and internal fluxes were calculated through flux optimization. Based on the internal flux analysis, glycolysis and pentose phosphate pathways were repressed under cultivation at pH 5, even though glutamate dehydrogenase increased GABA production. Notably, this repression was halted by adding succinic acid. Furthermore, proper sucA repression is a promising target for developing strains more capable of producing GABA.

Controlling enzyme inhibition using an expanded set of genetically encoded amino acids.

Enzyme inhibition plays an important role in drug development, metabolic pathway regulation, and biocatalysis with product inhibition. When an inhibitor has high structural similarities to the substrate of an enzyme, controlling inhibitor binding without affecting enzyme substrate binding is often challenging and requires fine-tuning of the active site. We hypothesize that an extended set of genetically encoded amino acids can be used to design an enzyme active site that reduces enzyme inhibitor binding without compromising substrate binding. As a model case, we chose murine dihydrofolate reductase (mDHFR), substrate dihydrofolate, and inhibitor methotrexate. Structural models of mDHFR variants containing non-natural amino acids complexed with each ligand were constructed to identify a key residue for inhibitor binding and non-natural amino acids to replace the key residue. Then, we discovered that replacing the key phenylalanine residue with two phenylalanine analogs (p-bromophenylalanine (pBrF) and L-2-naphthylalanine (2Nal)) enhances binding affinity toward the substrate dihydrofolate over the inhibitor by 4.0 and 5.8-fold, respectively. Such an enhanced selectivity is mainly due to a reduced inhibitor binding affinity by 2.1 and 4.3-fold, respectively. The catalytic efficiency of the mDHFR variant containing pBrF is comparable to that of wild-type mDHFR, whereas the mDHFR variant containing 2Nal exhibits a moderate decrease in the catalytic efficiency. The work described here clearly demonstrates the feasibility of selectively controlling enzyme inhibition using an expanded set of genetically encoded amino acids.

Different fates of Alzheimer's disease amyloid-ß fibrils remodeled by biocompatible small molecules.

Amyloid fibrils implicated in numerous human diseases are thermodynamically very stable. Stringent conditions that would not be possible in a physiological environment are often required to disrupt the stable fibrils. Recently, there is increasing evidence that small molecules can remodel amyloid fibrils in a physiologically relevant manner. In order to investigate possible fibril remodeling mechanisms using this approach, we performed comparative studies on the structural features of the different amyloid-ß (Aß) aggregates remodeled from Aß fibrils by three biocompatible small molecules: methylene blue; brilliant blue G; and erythrosine B. Combined with circular dichroism (CD), immuno-blotting, transmission electron microscopy (TEM), and atomic force microscopy (AFM) results, it was found that brilliant blue G- and erythrosine B-treatment generate fragmented Aß fibrils and protofibrils, respectively. In contrast, incubation of the Aß fibrils with methylene blue perturbs fibrillar structure, leading to amorphous Aß aggregates. Our findings provide insights on the molecular mechanism of amyloid fibril formation and remodeling and also illustrate the possibility of controlled changes in biomolecule nanostructures.

Real flue gas CO2 hydrogenation to formate by an enzymatic reactor using O2- and CO-tolerant hydrogenase and formate dehydrogenase.

It is challenging to capture carbon dioxide (CO2), a major greenhouse gas in the atmosphere, due to its high chemical stability. One potential practical solution to eliminate CO2 is to convert CO2 into formate using hydrogen (H2) (CO2 hydrogenation), which can be accomplished with inexpensive hydrogen from sustainable sources. While industrial flue gas could provide an adequate source of hydrogen, a suitable catalyst is needed that can tolerate other gas components, such as carbon monoxide (CO) and oxygen (O2), potential inhibitors. Our proposed CO2 hydrogenation system uses the hydrogenase derived from Ralstonia eutropha H16 (ReSH) and formate dehydrogenase derived from Methylobacterium extorquens AM1 (MeFDH1). Both enzymes are tolerant to CO and O2, which are typical inhibitors of metalloenzymes found in flue gas. We have successfully demonstrated that combining ReSH- and MeFDH1-immobilized resins can convert H2 and CO2 in real flue gas to formate via a nicotinamide adenine dinucleotide-dependent cascade reaction. We anticipated that this enzyme system would enable the utilization of diverse H2 and CO2 sources, including waste gases, biomass, and gasified plastics.