Development of growth selection system and pocket engineering of d-amino acid oxidase to enhance selective deamination activity toward d-phosphinothricin.

D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin (l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin (d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.

Enhancement of the substrate specificity of D-amino acid oxidase based on tunnel-pocket engineering.

D-Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of  D-amino acids, making it one of the most promising routes for synthesizing optically pure  L-amino acids, including  L-phosphinothricin ( L-PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate  D-PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward  D-PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M3rd -SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M3rd -SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M3rd -SHVG reshapes the tunnel-pocket and corrects the direction of enzyme-substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M3rd -SHVG as biocatalyst, 500 mM D, L-PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.

Antimicrobial D-amino acid oxidase-derived peptides specify gut microbiota.

The flavoenzyme D-amino acid oxidase (DAAO) is deputed to the degradation of D-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal D-amino acids to generate hydrogen peroxide (H2O2), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1-16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

Quantitative measurements of free and immobilized RgDAAO Michaelis-Menten constant using an electrochemical assay reveal the impact of covalent cross-linking on substrate specificity.

Challenges facing enzyme-based electrochemical sensors include substrate specificity, batch to batch reproducibility, and lack of quantitative metrics related to the effect of enzyme immobilization. We present a quick, simple, and general approach for measuring the effect of immobilization and cross-linking on enzyme activity and substrate specificity. The method can be generalized for electrochemical biosensors using an enzyme that releases hydrogen peroxide during its catalytic cycle. Using as proof of concept RgDAAO-based electrochemical biosensors, we found that the Michaelis-Menten constant (Km) decreases post immobilization, hinting at alterations in the enzyme kinetic properties and thus substrate specificity. We confirm the decrease in Km electrochemically by characterizing the substrate specificity of the immobilized RgDAAO using chronoamperometry. Our results demonstrate that enzyme immobilization affects enzyme substrate specificity and this must be carefully evaluated during biosensor development.

An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin.

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage.

PEG-DAAO conjugate: A promising tool for cancer therapy optimized by protein engineering.

The flavoenzyme D-amino acid oxidase (DAAO) represents a potentially good option for cancer enzyme prodrug therapy as it produces H2O2 using D-amino acids as substrates, compounds present at low concentration in vivo and that can be safely administered to regulate H2O2 production. We optimized the cytotoxicity of the treatment by: i) using an efficient enzyme variant active at low O2 and D-alanine concentrations (mDAAO); ii) improving the stability and half-life of mDAAO and the enhanced permeability and retention effect by PEGylation; and iii) inhibiting the antioxidant cellular system by a heme oxygenase-1 inhibitor (ZnPP). A very low amount of PEG-mDAAO (10 mU, 50 ng of enzyme) induces cytotoxicity on various tumor cell lines. Notably, PEG-mDAAO seems well suited for in vivo evaluation as it shows the same cytotoxicity at air saturation (21%) and 2.5% O2, a condition resembling the microenvironment found in the central part of tumors.

A detailed mechanism of the oxidative half-reaction of d-amino acid oxidase: another route for flavin oxidation.

d-Amino acid oxidase (DAAO) is a flavoenzyme whose inhibition is expected to have therapeutic potential in schizophrenia. DAAO catalyses hydride transfer from the substrate to the flavin in the reductive half-reaction, and the flavin is reoxidized by O2 in the oxidative half-reaction. Quantum mechanical/molecular mechanical calculations were performed and their results together with available experimental information were used to elucidate the detailed mechanism of the oxidative half-reaction. The reaction starts with a single electron transfer from FAD to O2, followed by triplet-singlet transition. FAD oxidation is completed by a proton coupled electron transfer to the oxygen species and the reaction terminates with H2O2 formation by proton transfer from the oxidized substrate to the oxygen species via a chain of water molecules. The substrate plays a double role by facilitating the first electron transfer and by providing a proton in the last step. The mechanism differs from the oxidative half-reaction of other oxidases.

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion.

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.

Synthesis and Biochemical Evaluation of Lid-Open D-Amino Acid Oxidase Inhibitors.

Most of the known inhibitors of D-amino acid oxidase (DAAO) are small polar molecules recognized by the active site of the enzyme. More recently a new class of DAAO inhibitors has been disclosed that interacts with loop 218-224 at the top of the binding pocket. These compounds have a significantly larger size and more beneficial physicochemical properties than most reported DAAO inhibitors, however, their structure-activity relationship is poorly explored. Here we report the synthesis and evaluation of this type of DAAO inhibitors that open the lid over the active site of DAAO. In order to collect relevant SAR data we varied two distinct parts of the inhibitors. A systematic variation of the pendant aromatic substituents according to the Topliss scheme resulted in DAAO inhibitors with low nanomolar activity. The activity showed low sensitivity to the substituents investigated. The variation of the linker connecting the pendant aromatic moiety and the acidic headgroup revealed that the interactions of the linker with the enzyme were crucial for achieving significant inhibitory activity. Structures and activities were analyzed based on available X-ray structures of the complexes. Our findings might support the design of drug-like DAAO inhibitors with advantageous physicochemical properties and ADME profile.

Human d-amino acid oxidase: The inactive G183R variant.

In the brain, the enzyme d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-serine, a main positive modulator of the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR). Dysregulation in d-serine signaling is implicated in the NMDAR dysfunctions observed in various brain diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia. A strain of ddY mice lacking DAAO activity due to the G181R substitution (DAAOG181R mice) and exhibiting increased d-serine concentration as compared to wild-type mice shows altered pain response, improved adaptative learning and cognitive functions, and larger hippocampal long-term potentiation. In past years, this mice line has been used to shed light on physiological and pathological brain functions related to NMDAR. Here, we decided to introduce the corresponding substitution in human DAAO (hDAAO). The recombinant G183R hDAAO is produced as an inactive apoprotein: the substitution alters the protein conformation that negatively affects the ability to bind the flavin cofactor in the orientation required for hydride-transfer during catalysis. At the cellular level, the overexpressed G183R hDAAO is not fully targeted to peroxisomes, forms protein aggregates showing a strong colocalization with ubiquitin, and significantly (7-fold) increases both the d-serine cellular concentration and the D/(D+L)-serine ratio. Taken together, our investigation warrants caution in using DAAOG181R mice: the abolition of enzymatic activity is coupled to DAAO aggregation, a central process in different pathological conditions. The effect due to G181R substitution in DAAO could be misleading: the effects due to impairment of d-serine degradation overlap with those related to aggregates accumulation.

Binding Residence Time through Scaled Molecular Dynamics: A Prospective Application to hDAAO Inhibitors.

Traditionally, a drug potency is expressed in terms of thermodynamic quantities, mostly Kd, and empirical IC50 values. Although binding affinity as an estimate of drug activity remains relevant, it is increasingly clear that it is also important to include (un)binding kinetic parameters in the characterization of potential drug-like molecules. Herein, we used standard in silico screening to identify a series of structurally related inhibitors of hDAAO, a flavoprotein involved in schizophrenia and neuropathic pain. We applied a novel methodology, based on scaled molecular dynamics, to rank them according to their residence times. Notably, we challenged the application in a prospective fashion for the first time. The good agreement between experimental residence times and the predicted residence times highlighted the procedure's reliability in both predictive and refinement scenarios. Additionally, through further inspection of the performed simulations, we substantiated a previous hypothesis on the involvement of a protein loop during ligand unbinding.

Immobilization of enzymes using a multifunctional fusion polypeptide.

OBJECTIVE: To design a fusion polypeptide combining functions of self-assembly and purification for immobilizing enzymes. RESULTS: A collagen-like polypeptide (CLP) was fused to an elastin-like polypeptide (ELP) through genetic engineering. CLP-ELP was separately fused to superoxide dismutase (SOD) and D-amino acid oxidase (DAAO). The recombinant enzymes were purified with using reversible phase transition. The interfering effect of H2O2 on the secondary structures of the recombinant enzymes was significantly reduced. The stability of the recombinant enzymes against denaturing by urea was improved. SOD-CLP-ELP exhibited a proteinaceous microporous network, and DAAO-CLP-ELP exhibited micro-clusters. The superoxide anion (•O2-) scavenging ability of SOD-CLP-ELP was 1.5 times that of SOD, and the catalytic efficiency of DAAO-CLP-ELP was 1.7 times that of DAAO. CONCLUSIONS: The advantages of the CLP-ELP-fused enzymes have been demonstrated and CLP-ELP can be used to immobilize other enzymes/proteins.

Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase.

The flavoprotein d-amino acid oxidase has long served as a paradigm for understanding the mechanism of oxidation of amino acids by flavoproteins. Recently, a mutant d-amino acid oxidase (Y228L/R283G) that catalyzed the oxidation of amines rather than amino acids was described [Yasukawa, K., et al. (2014) Angew. Chem., Int. Ed. 53, 4428-4431]. We describe here the use of pH and kinetic isotope effects with (R)-α-methylbenzylamine as a substrate to determine whether the mutant enzyme utilizes the same catalytic mechanism as the wild-type enzyme. The effects of pH on the steady-state and rapid-reaction kinetics establish that the neutral amine is the substrate, while an active-site residue, likely Tyr224, must be uncharged for productive binding. There is no solvent isotope effect on the kcat/Km value for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the kcat/Km value is pH-independent, with an average value of 5.3, similar to values found with amino acids as substrates for the wild-type enzyme and establishing that there is no commitment to catalysis with this substrate. The kcat/KO2 value is similar to that seen with amino acids as the substrate, consistent with the oxidative half-reaction being unperturbed by the mutation and with flavin oxidation preceding product release. All of the data are consistent with the mutant enzyme utilizing the same mechanism as the wild-type enzyme, transfer of hydride from the neutral amine to the flavin.

Altered dynamics upon oligomerization corresponds to key functional sites.

It is known that over half of the proteins encoded by most organisms function as oligomeric complexes. Oligomerization confers structural stability and dynamics changes in proteins. We investigate the effects of oligomerization on protein dynamics and its functional significance for a set of 145 multimeric proteins. Using coarse-grained elastic network models, we inspect the changes in residue fluctuations upon oligomerization and then compare with residue conservation scores to identify the functional significance of these changes. Our study reveals conservation of about ½ of the fluctuations, with » of the residues increasing in their mobilities and » having reduced fluctuations. The residues with dampened fluctuations are evolutionarily more conserved and can serve as orthosteric binding sites, indicating their importance. We also use triosephosphate isomerase as a test case to understand why certain enzymes function only in their oligomeric forms despite the monomer including all required catalytic residues. To this end, we compare the residue communities (groups of residues which are highly correlated in their fluctuations) in the monomeric and dimeric forms of the enzyme. We observe significant changes to the dynamical community architecture of the catalytic core of this enzyme. This relates to its functional mechanism and is seen only in the oligomeric form of the protein, answering why proteins are oligomeric structures. Proteins 2017; 85:1422-1434. © 2017 Wiley Periodicals, Inc.

An overview on D-amino acids.

More than half a century ago researchers thought that D-amino acids had a minor function compared to L-enantiomers in biological processes. Many evidences have shown that D-amino acids are present in high concentration in microorganisms, plants, mammals and humans and fulfil specific biological functions. In the brain of mammals, D-serine (D-Ser) acts as a co-agonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptors, responsible for learning, memory and behaviour. D-Ser metabolism is relevant for disorders associated with an altered function of the NMDA receptor, such as schizophrenia, ischemia, epilepsy and neurodegenerative disorders. On the other hand, D-aspartate (D-Asp) is one of the major regulators of adult neurogenesis and plays an important role in the development of endocrine function. D-Asp is present in the neuroendocrine and endocrine tissues and testes, and regulates the synthesis and secretion of hormones and spermatogenesis. Also food proteins contain D-amino acids that are naturally originated or processing-induced under conditions such as high temperatures, acid and alkali treatments and fermentation processes. The presence of D-amino acids in dairy products denotes thermal and alkaline treatments and microbial contamination. Two enzymes are involved in the metabolism of D-amino acids: amino acid racemase in the synthesis and D-amino acid oxidase in the degradation.

Elucidation of inhibitor-binding pockets of d-amino acid oxidase using docking simulation and N-sulfanylethylanilide-based labeling technology.

Because of the relevance of d-serine (d-Ser) to schizophrenia, inhibitors of d-amino acid oxidase (DAO), which catalyzes degradation of d-Ser in the presence of flavin adenine dinucleotide (FAD), are expected to be anti-schizophrenia therapeutics. In this study, binding pockets of DAO to its inhibitor 4-bromo-3-nitrobenzoic acid were searched by combining in silico docking simulation and labeling experiments employing an N-sulfanylethylanilide-based labeling technology that we have developed. The results clearly demonstrated that there are two binding pockets: one is shared with d-Ser and FAD, and the other is an unexpected cleft between the subunits of a DAO dimer. These findings will provide insight to aid the development of new DAO inhibitors. In addition, it was also proved that our labeling technology could be applicable to elucidate the binding pockets of proteins.

Novel insights into renal D-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo.

Chronic exposure to propiverine, a frequently prescribed pharmaceutical for treatment of overactive bladder and incontinence, provokes massive protein accumulation in the cytosol and nucleus of renal proximal tubule epithelial cells in rats. Previously, the accumulating protein was identified as D-amino acid oxidase (DAAO), a peroxisomal flavoenzyme expressed in kidney, liver and brain. The cellular mechanism of propiverine-induced DAAO accumulation, however, remains unexplained and poorly characterized. Therefore, to further increase the understanding of DAAO accumulation in rat kidney, this study aimed to characterize DAAO accumulations using differential immunofluorescent staining of rat kidney sections as well as in vitro binding analyses and proteasomal activity studies. We demonstrated that propiverine is neither a ligand of DAAO nor an inhibitor of the proteasome in vitro. However, propiverine treatment resulted in a significant decrease of peroxisomal size in rat proximal tubule epithelial cells. Moreover, peroxisomal catalase also accumulated in the cytosol and nuclei of propiverine-treated rats concurrently with DAAO. Taken together, our study indicates that propiverine treatment affects the trafficking and/or degradation of peroxisomal proteins such as DAAO and catalase by a so far unique and unknown mechanism.

Immobilization of D-Amino Acid Oxidase (DAAO) Enzyme on Hybrid Mesoporous MCF, SBA-15 and MCM-41 Nanomaterial.

Hybrid mesoporous materials as carriers for immobilization of D-amino acid oxidase (DAAO) were prepared via three steps: (i) hydrothermal synthesis of nanoporous MCF, SBA-15 and MCM-41 powders, (ii) functionalization with 3-aminopropyltriethoxysilane (APTES) by post-synthesis grafting; and (iii) activation with glutardialdehyde. The resulting mesostructured solids were characterized by various techniques: XRD, IR, TGA-DTA and N2 adsorption­desorption (BET). The characterization results indicated that these materials still maintained their structure after functionalization. IR data and TGA-DTA analysis demonstrated the existence of amine functional groups on the surface of APTES-functionalized samples. The DAAO immobilized on these materials exhibited higher catalytic activity and stability of enzyme for conversion of cephalosporin C (CPC) as compared to those of the non-functionalized ones. The catalytic activity and stability of enzyme decreased in the order MCF > SBA-15 > MCM-41.

Structure-function relationships in human d-amino acid oxidase variants corresponding to known SNPs.

In the brain, d-amino acid oxidase plays a key role in modulating the N-methyl-d-aspartate receptor (NMDAR) activation state, catalyzing the stereospecific degradation of the coagonist d-serine. A relationship between d-serine signaling deregulation, NMDAR dysfunction, and CNS diseases is presumed. Notably, the R199W substitution in human DAAO (hDAAO) was associated with familial amyotrophic lateral sclerosis (ALS), and further coding substitutions, i.e., R199Q and W209R, were also deposited in the single nucleotide polymorphism database. Here, we investigated the biochemical properties of these different hDAAO variants. The W209R hDAAO variant shows an improved d-serine degradation ability (higher activity and affinity for the cofactor FAD) and produces a greater decrease in cellular d/(d+l) serine ratio than the wild-type counterpart when expressed in U87 cells. The production of H2O2 as result of excessive d-serine degradation by this hDAAO variant may represent the factor affecting cell viability after stable transfection. The R199W/Q substitution in hDAAO altered the protein conformation and enzymatic activity was lost under conditions resembling the cellular ones: this resulted in an abnormal increase in cellular d-serine levels. Altogether, these results indicate that substitutions that affect hDAAO functionality directly impact on d-serine cellular levels (at least in the model cell system used). The pathological effect of the expression of the R199W hDAAO, as observed in familial ALS, originates from both protein instability and a decrease in kinetic efficiency: the increase in synaptic d-serine may be mainly responsible for the neurotoxic effect. This information is expected to drive future targeted treatments.

Let the substrate flow, not the enzyme: Practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions.

Exploiting enzymes for chemical synthesis in flow microreactors necessitates their reuse for multiple rounds of conversion. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach, but practical methods for it are lacking. Using fusion to a silica-binding module to engineer enzyme adsorption to glass surfaces, we show convenient immobilization of d-amino acid oxidase on borosilicate microchannel plates. In confocal laser scanning microscopy, channel walls appeared uniformly coated with target protein. The immobilized enzyme activity was in the range expected for monolayer coverage of the plain surface with oxidase (2.37 × 10(-5) nmol/mm(2) ). Surface attachment of the enzyme was completely stable under flow. The operational half-life of the immobilized oxidase (25°C, pH 8.0; soluble catalase added) was 40 h. Enzymatic oxidation of d-Met into α-keto-γ-(methylthio)butyric acid was characterized in single-pass and recycle reactor configurations, employing in-line measurement of dissolved O2 , and off-line determination of the keto-acid product. Reaction-diffusion time-scale analysis for different flow conditions showed that the heterogeneously catalyzed reaction was always slower than diffusion of O2 to the solid surface (DaII ≤ 0.3). Potential of the microreactor for intensifying O2 -dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342-2349. © 2016 Wiley Periodicals, Inc.