Development of an Enzyme Cascade System for the Synthesis of Enantiomerically Pure D-Amino Acids Utilizing Ancestral L-Amino Acid Oxidase.

Enantiomerically pure D-amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide-based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L-amino acids into their D-isomers. The system integrates four enzymes: ancestral L-amino acid oxidase (AncLAAO-N4), D-amino acid dehydrogenase (DAADH), D-glucose dehydrogenase (GDH), and catalase. AncLAAO-N4 initiates the process by converting L-amino acids to corresponding keto acids, which are then stereo-selectively aminated to D-amino acids by DAADH using NADPH and NH4Cl. Concurrently, any generated H2O2 is decomposed into O2 and H2O by catalase, while GDH regenerates NADPH from D-glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D-amino acids, including a D-Phe derivative, three D-Trp derivatives, and D-phenylglycine, all with high enantiopurity (>99 % ee) at a preparative scale (>100 mg). This system demonstrates a versatile approach for producing a diverse array of D-amino acids.

Effect of eribulin on epithelial-mesenchymal transition plasticity in metastatic breast cancer: An exploratory, prospective study.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer metastasis and treatment resistance, which worsens prognosis. In phase III trials, eribulin improved overall survival in metastatic breast cancer (MBC) patients. In preclinical studies, eribulin suppressed EMT. However, clinical data on the use of eribulin for MBC patients are limited. In this exploratory, prospective study, we examined the effect of eribulin on EMT in MBC patients. Twenty-two patients aged 44-82 years with recurrent breast cancer or MBC were treated with eribulin. Breast cancer tissue samples were obtained before treatment and on Day 15 ± 5 of the first cycle of eribulin treatment. EMT markers (E-cadherin, claudin-3, vimentin, and N-cadherin) were analyzed using western blotting. EMT changes were evaluated based on the ratio of epithelial to mesenchymal markers before and after treatment in individual tumors. E-cadherin/vimentin, claudin-3/vimentin, E-cadherin/N-cadherin, and claudin-3/N-cadherin ratios were significantly higher after treatment (p = .007, p = .005, p = .006, and p = .011, respectively). Based on E-cadherin/vimentin, 65.0% of tumors shifted to an epithelial phenotype, as compared to 66.7% based on claudin-3/vimentin, 84.6% based on E-cadherin/N-cadherin, and 71.4% based on claudin-3/N-cadherin ratios. Thus, our results showed that eribulin suppressed EMT in breast cancer tissues.

Comprehensive genomic evaluation of advanced and recurrent breast cancer patients for tailored precision treatments.

AIM: The aim of this study was to investigate genetic alterations within breast cancer in the setting of recurrent or de novo stage IV disease. PATIENTS AND METHODS: This study included 22 patients with recurrent breast cancer (n = 19) and inoperable de novo stage IV breast cancer (n = 3). For next generation sequencing, FoundationOneCDx (F1CDx) (Foundation Medicine Inc., Cambridge, MA, USA) was performed in 21 patients and FoundationOneLiquid CDx was performed in 1 patient. RESULTS: Median age was 62.9 years (range, 33.4-82.1). Pathological diagnoses of specimens included invasive ductal carcinoma (n = 19), invasive lobular carcinoma (n = 2), and invasive micropapillary carcinoma (n = 1). F1CDx detected a median of 4.5 variants (range, 1-11). The most commonly altered gene were PIK3CA (n = 9), followed by TP53 (n = 7), MYC (n = 4), PTEN (n = 3), and CDH1 (n = 3). For hormone receptor-positive patients with PIK3CA mutations, hormonal treatment plus a phosphoinositide 3-kinase inhibitor was recommended as the treatment of choice. Patients in the hormone receptor-negative and no human epidermal growth factor receptor 2 expression group had significantly higher tumor mutational burden than patients in the hormone receptor-positive group. A BRCA2 reversion mutation was revealed by F1CDx in a patient with a deleterious germline BRCA2 mutation during poly ADP ribose polymerase inhibitor treatment. CONCLUSION: Guidance on tailored precision therapy with consideration of genomic mutations was possible for some patients with information provided by F1CDx. Clinicians should consider using F1CDx at turning points in the course of the disease.

Definition of an Index Parameter To Screen Highly Functional Enzymes Derived from a Biochemical and Thermodynamic Analysis of Ancestral meso-Diaminopimelate Dehydrogenases.

Sequence-based protein design approaches are being adopted to generate highly functional enzymes; however, screening the enzymes remains a time-consuming task. In this study, by analyzing the enzymatic properties of four ancestral meso-2,6-diaminopimelate dehydrogenases (AncDAPDHs), AncDAPDH-N1, -N2, -N3, and -N4, we attempted to define a new index parameter that is helpful for efficiently screening the enzymes. Biochemical and thermodynamic analyses indicated that only AncDAPDH-N4 exhibited greater thermal stability than and activity similar to those of native DAPDHs. Structural and sequence comparisons between DAPDH from Corynebacterium glutamicum (CgDAPDH) and the AncDAPDHs suggested that "quality of mutations" is a potential index parameter. In fact, the mutations introduced from CgDAPDH to AncDAPDH-N4 correlated highly with the mutations accumulated during the evolution process from mesophiles to thermophiles. These results suggest that, although there are several exceptions, the correlation coefficient can be used as an index parameter for screening high-functioning enzymes from sequence data.

Direct evaluation of polarity of the ligand binding pocket in retinoid X receptor using a fluorescent solvatochromic agonist.

High selectivity of small-molecule drug candidates for their target molecule is important to minimize potential side effects. One factor that contributes to the selectivity is the internal polarity of the ligand-binding pocket (LBP) in the target molecule, but this is difficult to measure. Here, we first confirmed that the retinoid X receptor (RXR) agonist 6-(ethyl(1-isobutyl-2-oxo-4-(trifluoromethyl)-1,2-dihydroquinolin-7-yl)amino)nicotinic acid (NEt-iFQ, 1) exhibits fluorescence solvatochromism, i.e., its Stokes shift depends on the polarity of the solvent, and then we utilized this property to directly measure the internal polarity of the RXRα-LBP. The Stokes shift of 1 when bound to the RXRα-LBP corresponded to that of 1 in chloroform solution. This finding is expected to be helpful for designing RXR-selective ligands. A similar approach should be appliable to evaluate the internal polarity of the LBPs of other receptors.

Clinical performance of digital breast tomosynthesis-guided vacuum-assisted biopsy: a single-institution experience in Japan.

BACKGROUND: The purpose of this study was to evaluate the clinical performance of Digital Breast Tomosynthesis guided vacuum-assisted biopsy (DBT-VAB) for microcalcifications in the breast. METHODS: Retrospective review of 131 mammography-guided VABs at our institution were performed. All of the targets were calcification lesion suspicious for cancer. 45 consecutive stereotactic vacuum-assisted biopsies (ST-VABs) and 86 consecutive DBT-VABs were compared. Written informed consent was obtained. Tissue sampling methods and materials were the same with both systems. Student's t-test was used to compare procedure time and the Fisher's exact test was used to compare success rate, complications, and histopathologic findings for the 2 methods. RESULTS: The tissue sampling success rate was 95.6% for ST-VAB (43/45) and 97.7% (84/86) for DBT-VAB. Time for positioning (10.6 ± 6.4 vs. 6.7 ± 5.3 min), time for biopsy (33.4 ± 13.1 vs. 22.5 ± 13.1 min), and overall procedure time (66.6 ± 16.6 min vs. 54.5 ± 13.0 min) were substantially shorter with DBT-VAB (P < 0.0001). There were no differences in the distribution of pathological findings between the 2 groups. CONCLUSION: Depth information and stable visibility of the target provided by DBT images led to quick decisions about target coordinates and improved the clinical performance of microcalcification biopsies.

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining.

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the kcat/Km value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.

Design of a Full-Consensus Glutamate Decarboxylase and Its Application to GABA Biosynthesis.

Glutamate decarboxylase (GAD) catalyses the decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Improvement of the enzymatic properties of GAD is important for the low-cost synthesis of GABA. In this study, utilizing sequences of enzymes homologous with GAD from lactic acid bacteria, highly mutated GADs were designed using sequence-based protein design methods. Two mutated GADs, FcGAD and AncGAD, generated by full-consensus design and ancestral sequence reconstruction, had more desirable properties than native GADs. With respect to thermal stability, the half-life of the designed GADs was about 10 °C higher than that of native GAD. The productivity of FcGAD was considerably higher than those of known GADs; more than 250 mg/L of purified enzyme could be produced in the E. coli expression system. In a production test using 26.4 g of l-glutamate and 3.0 g of resting cells, 17.2 g of GABA could be prepared within one hour, without purification, in a one-pot synthesis.

Enzymatic Conjugation of Modified RNA Fragments by Ancestral RNA Ligase AncT4_2.

Oligonucleotide therapeutics have great potential as a next-generation approach to treating intractable diseases. Large quantities of modified DNA/RNA containing xenobiotic nucleic acids (XNAs) must be synthesized before clinical application. In this study, the ancestral RNA ligase AncT4_2 was designed by ancestral sequence reconstruction (ASR) to perform the conjugation reaction of modified RNA fragments. AncT4_2 had superior properties to native RNA ligase 2 from T4 phage (T4Rnl2), including high productivity, a >2.5-fold-higher turnover number, and >10°C higher thermostability. One remarkable point is the broad substrate selectivity of AncT4_2; the activity of AncT4_2 toward 17 of the modified RNA fragments was higher than that of T4Rnl2. The activity was estimated by measuring the conjugation reaction of two RNA strands, 3'-OH (12 bp) and 5'-PO4 (12 bp), in which the terminal and penultimate positions of the 3'-OH fragment and the first and second positions of the 5'-PO4 fragment were substituted by 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-H, respectively. The enzymatic properties of AncT4_2 allowed the enzyme to conjugate large quantities of double-stranded RNA coding for patisiran (>400 µM level), which was formed by four RNA fragments containing 2'-OMe-substituted nucleic acids. Structural analysis of modeled AncT4_2 suggested that protein dynamics were changed by mutation to Gly or indel during ASR and that this may positively impact the conjugation of modified RNA fragments with the enzyme. AncT4_2 is expected to be a key biocatalyst in synthesizing RNA therapeutics by an enzymatic reaction. IMPORTANCE RNA therapeutics is one of the next-generation medicines for treating various diseases. Our designed ancestral RNA ligase AncT4_2 exhibited excellent enzymatic properties, such as high thermal stability, productivity, specific activity, and broad substrate selectivity compared to native enzymes. These advantages create the potential for AncT4_2 to be applied in conjugating the modified RNA fragments containing various xenobiotic nucleic acids. In addition, patisiran, a known polyneuropathy therapeutic, could be synthesized from four fragmented oligonucleotides at a preparative scale. Taken together, these findings indicate AncT4_2 could open the door to synthesizing RNA therapeutics by enzymatic reaction at large-scale production.

Airway obstruction requiring tracheal intubation after fine-needle aspiration of the thyroid gland: a case report.

BACKGROUND: There have been no reports of tracheal intubation for airway obstruction after acute thyroid swelling following fine-needle aspiration (FNA) of the thyroid gland. CASE PRESENTATION: A 58-year-old woman with a 22 mm × 13 mm right hypervascular thyroid nodule underwent FNA once with a 22G needle under ultrasonographic guidance. Shortly after the aspiration, ultrasound revealed hypoechoic swelling with a crack-like pattern. The patient was observed under bed rest in the Fowler position and received intravenous steroids. A computed tomography (CT) scan showed swelling not only of the thyroid but also of the retropharyngeal space, and the patient complained of difficulty swallowing saliva. Laryngeal fiberscopy revealed protrusion of the posterior pharyngeal wall, edematous changes in the mucosa of the pharynx and epiglottis, and retention of saliva. The patient was intubated awake and hydrocortisone was administered every 8 h. She was extubated 3 days after FNA and discharged without any complications. CONCLUSIONS: When neck swelling is noticed after FNA, ultrasonographic findings are especially important to assess potential causes. If airway obstruction is suspected, CT findings and fiberscope observation of the pharynx provide particularly useful information.

Partial Consensus Design and Enhancement of Protein Function by Secondary-Structure-Guided Consensus Mutations.

Consensus design (CD) is a representative sequence-based protein design method that enables the design of highly functional proteins by analyzing vast amounts of protein sequence data. This study proposes a partial consensus design (PCD) of a protein as a derivative approach of CD. The method replaces the target protein sequence with a consensus sequence in a secondary-structure-dependent manner (i.e., regionally dependent and divided into α-helix, ß-sheet, and loop regions). In this study, we generated several artificial partial consensus l-threonine 3-dehydrogenases (PcTDHs) by PCD using the TDH from Cupriavidus necator (CnTDH) as a target protein. Structural and functional analysis of PcTDHs suggested that thermostability would be independently improved when consensus mutations are introduced into the loop region of TDHs. On the other hand, enzyme kinetic parameters (kcat/Km) and average productivity would be synergistically enhanced by changing the combination of the mutations-replacement of one region of CnTDH with a consensus sequence provided only negative effects, but the negative effects were nullified when the two regions were replaced simultaneously. Taken together, we propose the hypothesis that there are protein regions that encode individual protein properties, such as thermostability and activity, and that the introduction of consensus mutations into these regions could additively or synergistically modify their functions.

Fluorescence properties of retinoid X receptor antagonist NEt-SB.

Ligands of retinoid X receptors (RXRs) are effective against various diseases, so there is a need for efficient screening methods to discover new ligands. Existing screening methods are complex and time-consuming, and a simple fluorescence assay would be highly desirable. Here, we focused on NEt-SB (4), which has a stilbene structure, as a candidate for this purpose, and examined its fluorescence properties in detail. The fluorescence intensity of 4 was remarkably increased in highly viscous solvents and upon binding to hRXRα-LBD, due to suppression of free rotation of the stilbene moiety. Although the relatively low fluorescence intensity and the short fluorescence wavelength of 4 make this compound itself unsuitable for use in RXR binding assay, our findings provide a basis for further structural evolution, which may lead to a derivative that would be suitable for fluorescence assay of RXR binders.

Trp-Trp acts as a multifunctional blocker for human bitter taste receptors, hTAS2R14, hTAS2R16, hTAS2R43, and hTAS2R46.

Many functional food ingredients activate human bitter taste receptors (hTAS2Rs). In this study, A novel inhibitor, Trp-Trp, for hTAS2R14 was identified by searching for the agonist peptide's analogs. Trp-Trp also inhibited hTAS2R16, hTAS2R43, and hTAS2R46, which share the same agonists with hTAS2R14. The multifunctional characteristic of Trp-Trp is advantageous for use as bitterness-masking agents in functional foods.

Computational Analysis Reveals a Critical Point Mutation in the N-Terminal Region of the Signaling Lymphocytic Activation Molecule Responsible for the Cross-Species Infection with Canine Distemper Virus.

Infection of hosts by morbilliviruses is facilitated by the interaction between viral hemagglutinin (H-protein) and the signaling lymphocytic activation molecule (SLAM). Recently, the functional importance of the n-terminal region of human SLAM as a measles virus receptor was demonstrated. However, the functional roles of this region in the infection process by other morbilliviruses and host range determination remain unknown, partly because this region is highly flexible, which has hampered accurate structure determination of this region by X-ray crystallography. In this study, we analyzed the interaction between the H-protein from canine distemper virus (CDV-H) and SLAMs by a computational chemistry approach. Molecular dynamics simulations and fragment molecular orbital analysis demonstrated that the unique His28 in the N-terminal region of SLAM from Macaca is a key determinant that enables the formation of a stable interaction with CDV-H, providing a basis for CDV infection in Macaca. The computational chemistry approach presented should enable the determination of molecular interactions involving regions of proteins that are difficult to predict from crystal structures because of their high flexibility.

Protein Sequence Selection Method That Enables Full Consensus Design of Artificial l-Threonine 3-Dehydrogenases with Unique Enzymatic Properties.

Exponentially increasing protein sequence data enables artificial enzyme design using sequence-based protein design methods, including full-consensus protein design (FCD). The success of artificial enzyme design is strongly dependent on the nature of the sequences used. Hence, sequences must be selected from databases and curated libraries prepared to enable a successful design by FCD. In this study, we proposed a selection approach regarding several key residues as sequence motifs. We used l-threonine 3-dehydrogenase (TDH) as a model to test the validity of this approach. In the classification, four residues (143, 174, 188, and 214) were used as key residues. We classified thousands of TDH homologous sequences into five groups containing hundreds of sequences. Utilizing sequences in the libraries, we designed five artificial TDHs by FCD. Among the five, we successfully expressed four in soluble form. Biochemical analysis of artificial TDHs indicated that their enzymatic properties vary; half of the maximum measured enzyme activity (t1/2) and activation energies were distributed from 53 to 65 °C and from 38 to 125 kJ/mol, respectively. The artificial TDHs had unique kinetic parameters, distinct from one another. Structural analysis indicates that consensus mutations are mainly introduced in the secondary or outer shell. The functional diversity of the artificial TDHs is due to the accumulation of mutations that affect their physicochemical properties. Taken together, our findings indicate that our proposed approach can help generate artificial enzymes with unique enzymatic properties.

Elucidation of Molecular Mechanism of a Selective PPARα Modulator, Pemafibrate, through Combinational Approaches of X-ray Crystallography, Thermodynamic Analysis, and First-Principle Calculations.

The selective PPARα modulator (SPPARMα) is expected to medicate dyslipidemia with minimizing adverse effects. Recently, pemafibrate was screened from the ligand library as an SPPARMα bearing strong potency. Several clinical pieces of evidence have proved the usefulness of pemafibrate as a medication; however, how pemafibrate works as a SPPARMα at the molecular level is not fully known. In this study, we investigate the molecular mechanism behind its novel SPPARMα character through a combination of approaches of X-ray crystallography, isothermal titration calorimetry (ITC), and fragment molecular orbital (FMO) analysis. ITC measurements have indicated that pemafibrate binds more strongly to PPARα than to PPARγ. The crystal structure of PPARα-ligand binding domain (LBD)/pemafibrate/steroid receptor coactivator-1 peptide (SRC1) determined at 3.2 Å resolution indicates that pemafibrate binds to the ligand binding pocket (LBP) of PPARα in a Y-shaped form. The structure also reveals that the conformation of the phenoxyalkyl group in pemafibrate is flexible in the absence of SRC1 coactivator peptide bound to PPARα; this gives a freedom for the phenoxyalkyl group to adopt structural changes induced by the binding of coactivators. FMO calculations have indicated that the accumulation of hydrophobic interactions provided by the residues at the LBP improve the interaction between pemafibrate and PPARα compared with the interaction between fenofibrate and PPARα.

Following the Evolutionary Track of a Highly Specific l-Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes.

Following the evolutionary track of enzymes can help elucidate how enzymes attain their characteristic functions, such as thermal adaptation and substrate selectivity, during the evolutionary process. Ancestral sequence reconstruction (ASR) is effective for following evolutionary processes if sufficient sequence data are available. Selecting sequences from the data to generate a curated sequence library is necessary for the successful design of artificial proteins by ASR. In this study, we tried to follow the evolutionary track of l-arginine oxidase (AROD), a flavin adenine dinucleotide (FAD)-dependent amino acid oxidase (LAAO) that exhibits high specificity for l-arginine. The library was generated by selecting sequences in which the 15th, 50th, 332nd, and 580th residues are Gly, Ser, Trp, and Thr, respectively. We excluded sequences that are either extremely short or long and those with a low degree of sequence identity. Three ancestral ARODs (AncARODn0, AncARODn1, and AncARODn2) were designed using the library. Subsequently, we expressed the ancestral ARODs as well as native Oceanobacter kriegii AROD (OkAROD) in bacteria. AncARODn0 is phylogenetically most remote from OkAROD, whereas AncARODn2 is most similar to OkAROD. Thermal stability was gradually increased by extending AROD sequences back to the progenitor, while the temperature at which the residual activity is half of the maximum measured activity (T1/2) of AncARODn0 was >20°C higher than that of OkAROD. Remarkably, only AncARODn0 exhibited broad substrate selectivity similar to that of conventional promiscuous LAAO. Taken together, our findings led us to infer that AROD may have evolved from a highly thermostable and promiscuous LAAO.IMPORTANCE In this study, we attempted to infer the molecular evolution of a recently isolated FAD-dependent l-arginine oxidase (AROD) that oxidizes l-arginine to 2-ketoarginine. Utilizing 10 candidate AROD sequences, we obtained a total of three ancestral ARODs. In addition, one native AROD was obtained by cloning one of the candidate ARODs. The candidate sequences were selected utilizing a curation method defined in this study. All the ARODs were successfully expressed in Escherichia coli for analysis of their biochemical functions. The catalytic activity of our bacterially expressed ancestral ARODs suggests that our ASR was successful. The ancestral AROD that is phylogenetically most remote from a native AROD has the highest thermal stability and substrate promiscuity. Our findings led us to infer that AROD evolved from a highly thermostable and promiscuous LAAO. As an application, we can design artificial ARODs with improved functions compared with those of native ones.

Development of an Analysis Toolkit, AnalysisFMO, to Visualize Interaction Energies Generated by Fragment Molecular Orbital Calculations.

In modern praxis, a knowledge-driven design of pharmaceutical compounds relies heavily on protein structure data. Nonetheless, quantification of the interaction between protein and ligand is of great importance in the theoretical evaluation of the ability of a pharmaceutical compound to comply with certain expectations. The FMO (fragment molecular orbital) method is handy in this regard. However, the physical complexity and the number of the interactions within a protein-ligand complex renders analysis of the results somewhat complicated. This situation prompted us to develop the 3D-visualization of interaction energies in protein (3D-VIEP) method; the toolkit AnalysisFMO, which should enable a more efficient and convenient workflow with FMO data generated by quantum-chemical packages such as GAMESS, PAICS, and ABINIT-MP. AnalysisFMO consists of two separate units, RbAnalysisFMO, and the PyMOL plugins. The former can extract interfragment interaction energies (IFIEs) or pair interaction energies (PIEs) from the FMO output files generated by the aforementioned quantum-chemical packages. The PyMOL plugins enable visualization of IFIEs or PIEs in the protein structure in PyMOL. We demonstrate the use of this tool on a lectin protein from Burkholderia cenocepacia in which FMO analysis revealed the existence of a new interaction between Gly84 and fucose. Moreover, we found that second-shell interactions are crucial in forming the sugar binding site. In the case of bilirubin oxidase from Myrothecium verrucaria (MvBO), we predict that interactions between Asp105 and three His residues (His401, His403, and His136) are essential for optimally positioning the His residues to coordinate Cu atoms to form one Type 2 and two Type 3 Cu ions.

Benchmark Analysis of Native and Artificial NAD+-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data.

The expansion of protein sequence databases has enabled us to design artificial proteins by sequence-based design methods, such as full-consensus design (FCD) and ancestral-sequence reconstruction (ASR). Artificial proteins with enhanced activity levels compared with native ones can potentially be generated by such methods, but successful design is rare because preparing a sequence library by curating the database and selecting a method is difficult. Utilizing a curated library prepared by reducing conservation energies, we successfully designed two artificial l-threonine 3-dehydrogenases (SDR-TDH) with higher activity levels than native SDR-TDH, FcTDH-N1, and AncTDH, using FCD and ASR, respectively. The artificial SDR-TDHs had excellent thermal stability and NAD+ recognition compared to native SDR-TDH from Cupriavidus necator (CnTDH); the melting temperatures of FcTDH-N1 and AncTDH were about 10 and 5 °C higher than that of CnTDH, respectively, and the dissociation constants toward NAD+ of FcTDH-N1 and AncTDH were 2- and 7-fold lower than that of CnTDH, respectively. Enzymatic efficiency of the artificial SDR-TDHs were comparable to that of CnTDH. Crystal structures of FcTDH-N1 and AncTDH were determined at 2.8 and 2.1 Å resolution, respectively. Structural and MD simulation analysis of the SDR-TDHs indicated that only the flexibility at specific regions was changed, suggesting that multiple mutations introduced in the artificial SDR-TDHs altered their flexibility and thereby affected their enzymatic properties. Benchmark analysis of the SDR-TDHs indicated that both FCD and ASR can generate highly functional proteins if a curated library is prepared appropriately.

Improvement of ST0452 N-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering.

We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibited over 4 times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity.IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes.