Multifunctionality of a low-specificity L-threonine aldolase from the hyperthermophile Thermotoga maritima.

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual D-lysine in addition to the typical D-alanine and D-glutamate. Previously, we identified the D-lysine and D-glutamate biosynthetic pathways of T. maritima. Additionally, we reported some multifunctional enzymes involved in amino acid metabolism. In the present study, we characterized the enzymatic properties of TM1744 (threonine aldolase) to probe both its potential multifunctionality and D-amino acid metabolizing activities. TM1744 displayed aldolase activity toward both L-allo-threonine and L-threonine, and exhibited higher activity toward L-threo-phenylserine. It did not function as an aldolase toward D-allo-threonine or D-threonine. Furthermore, TM1744 had racemase activity toward two amino acids, although its racemase activity was lower than its aldolase activity. TM1744 did not have other amino acid metabolizing activities. Therefore, TM1744 is a low-specificity L-threonine aldolase with limited racemase activity.

Characterisation of a microelectrochemical biosensor for real-time detection of brain extracellular d-serine.

A modified development protocol and concomitant characterisation of a first generation biosensor for the detection of brain extracellular d-serine is reported. Functional parameters important for neurochemical monitoring, including sensor sensitivity, O2 interference, selectivity, shelf-life and biocompatibility were examined. Construction and development involved the enzyme d-amino acid oxidase (DAAO), utilising a dip-coating immobilisation method employing a new extended drying approach. The resultant Pt-based polymer enzyme composite sensor achieved high sensitivity to d-serine (0.76 ± 0.04 nA mm-2. µM-1) and a low µM limit of detection (0.33 ± 0.02 µM). The in-vitro response time was within the solution stirring time, suggesting potential sub-second in-vivo response characteristics. Oxygen interference studies demonstrated a 1 % reduction in current at 50 µM O2 when compared to atmospheric O2 levels (200 µM), indicating that the sensor can be used for reliable neurochemical monitoring of d-serine, free from changes in current associated with physiological O2 fluctuations. Potential interference signals generated by the principal electroactive analytes present in the brain were minimised by using a permselective layer of poly(o-phenylenediamine), and although several d-amino acids are possible substrates for DAAO, their physiologically relevant signals were small relative to that for d-serine. Additionally, changing both temperature and pH over possible in vivo ranges (34-40 °C and 7.2-7.6 respectively) resulted in no significant effect on performance. Finally, the biosensor was implanted in the striatum of freely moving rats and used to monitor physiological changes in d-serine over a two-week period.

A dual-function selection system enables positive selection of multigene CRISPR mutants and negative selection of Cas9-free progeny in Arabidopsis.

The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants. However, screening edited alleles, particularly those with multiplex editing, from herbicide- or antibiotic-resistant transgenic plants and segregating out the Cas9 transgene represent two laborious processes. Current solutions to facilitate these processes rely on different selection markers. Here, by taking advantage of the opposite functions of a d-amino acid oxidase (DAO) in detoxifying d-serine and in metabolizing non-toxic d-valine to a cytotoxic product, we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of Cas9-containing progeny in Arabidopsis thaliana. Among five DAOs tested in Escherichia coli, the one encoded by Trigonopsis variabilis (TvDAO) could confer slightly stronger d-serine resistance than other homologs. Transgenic expression of TvDAO in Arabidopsis allowed a clear distinction between transgenic and non-transgenic plants in both d-serine-conditioned positive selection and d-valine-conditioned negative selection. As a proof of concept, we combined CRISPR-induced single-strand annealing repair of a dead TvDAO with d-serine-based positive selection to help identify transgenic plants with multiplex editing, where d-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin. Subsequently, d-valine-based negative selection successfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inherited mutations. Collectively, this work provides a novel strategy to ease CRISPR mutant identification and Cas9 transgene elimination using a single selection marker, which promises more efficient and simplified multiplex CRISPR editing in plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00132-6.

Enhancing the amination activity of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by modifying the crucial residue His154 for deamination.

During the deamination and amination processes of meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH), residue R71 was observed to display distinct functions. H154 has been proposed as a basic residue that facilitates water molecules to attack the D-chiral carbon of meso-DAP during deamination. Inspired by the phenomenon of R71, the effects of H154 during deamination and amination were investigated in this study with the goal of enhancing the amination activities of StDAPDH. Single site saturation mutagenesis indicated that almost all of the H154 mutants completely lost their deamination activity towards meso-DAP. However, some H154 variants showed enhanced kcat/Km values towards pyruvic acid and other bulky 2-keto acids, such as 2-oxovaleric acid, 4-methyl-2-oxopentanoic acid, 2-ketobutyric acid, and 3-methyl-2-oxobutanoic acid. When combined with the previously reported W121L/H227I mutant, triple mutants with significantly improved kcat/Km values (2.4-, 2.5-, 2.5-, and 4.0-fold) towards these 2-keto acids were obtained. Despite previous attempts, mutations at the H154 site did not yield the desired results. Moreover, this study not only recognizes the distinctive impact of H154 on both the deamination and amination reactions, but also provides guidance for further high-throughput screening in protein engineering and understanding the catalytic mechanism of StDAPDH.

Through virtual saturation mutagenesis and rational design for superior substrate conversion in engineered d-amino acid oxidase.

The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure.

Simultaneous analysis of DL-Amino acids in foods and beverages using a highly sensitive chiral resolution labeling reagent.

Amino acids with various functions are abundant in living organisms and foods. Recent advances in analytical technology show that trace amounts of D-amino acids exist in living organisms and foods. In addition, studies show that these amino acids are involved in various physiological functions that differ from those of L-amino acids. Thus, a technique for analyzing DL-amino acids is required. However, the simultaneous separation and highly sensitive detection of DL-amino acids are complicated; therefore, highly sensitive analytical methods that can rapidly separate and identify compounds are required. We previously developed our original chiral resolution labeling reagents for the separation and highly sensitive detection of DL-amino acids. Here, we developed a simple method for the rapid separation and highly sensitive detection of DL-amino acids in various foods and beverages by liquid chromatography-mass spectrometry (LC-MS) using an octadecyl (C18) column after labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamineamide (D-FDLDA; enantiomeric excess > 99.9 %). In addition, we synthesized a stable isotope (13C6)-labeled D-FDLDA (13C6-D-FDLDA) and established an analytical method that can accurately identify the peak of each DL-amino acid. MS sensitivity of DL-amino acids labeled with our labeling reagent was higher than that of conventional labeling reagents (Marfey's reagents). The labeling reagent was neither desorbed from each DL-amino acid nor degraded for at least 1 week at 4 °C. Furthermore, we determined the DL-amino acid contents in foods and beverages using the proposed method, and differences in the total amino acid content and D/L ratio in each food and beverage were observed.

A novel series of metazoan L/D peptide isomerases.

The function of endogenous cell-cell signaling peptides relies on their interactions with cognate receptors, which in turn are influenced by the peptides' structures, necessitating a comprehensive understanding of the suite of post-translational modifications of the peptide. Herein, we report the initial characterization of putative peptide isomerase enzymes extracted from R. norvegicus, A. californica, and B. taurus tissues. These enzymes are both tissue and substrate-specific across all three organisms. Notably, the lungs of the mammalian species, and the central nervous system of the mollusk displayed the highest isomerase activity among the examined tissues. In vitro enzymatic conversion was observed for several endogenous peptides, such as the tetrapeptide GFFD in A. californica, and mammalian neuropeptide FF in R. norvegicus and B. taurus. To understand their mode of action, we explored the effects of several inhibitors on these enzymes, which suggest common active site residues. While further characterization of these enzymes is required, the investigations emphasize a widespread and overlooked enzyme activity related to the creation of bioactive peptides.

Urinary D-amino acid profiles in cats with chronic kidney disease.

Chronic kidney disease (CKD) is highly prevalent in domestic cats. This study aimed to compare urinary D-amino acid levels between control and CKD-afflicted cats as a novel noninvasive method for assessing CKD. Cats were divided into control and CKD stage II groups in accordance with the International Renal Interest Society guidelines. The urinary DL-amino acid levels of the cats were analyzed using chiral tandem liquid chromatography-tandem mass spectrometry, and their medical records were investigated. The CKD group had considerably lower urinary D-amino acid concentrations and enantiomeric ratios than the control group. The total urinary D-amino acid contents significantly correlated with blood parameters (creatinine and urea nitrogen). These findings may contribute towards the detection of CKD stage II in domestic cats.

INTERACT: a randomized phase 2 study of the DAAO inhibitor luvadaxistat in adults with schizophrenia.

Deficits in N-methyl-d-aspartate receptor (NMDAR) signaling are implicated in the pathogenesis of schizophrenia. Luvadaxistat (TAK-831/NBI-1065844) is an investigational d-amino acid oxidase (DAAO) inhibitor that increases d-serine levels at NMDAR coagonist sites. INTERACT is a phase 2 randomized, placebo-controlled study that evaluated the efficacy and safety of three doses of luvadaxistat, covering a range of DAAO occupancy and d-serine levels, in patients with schizophrenia with persistent negative symptoms. The study included a 14-day, single-blinded placebo run-in period and a 12-week, double-blinded treatment period. The primary efficacy endpoint was the 12-week change from baseline in Positive and Negative Syndrome Scale-Negative Symptom Factor Score (PANSS NSFS). Secondary efficacy endpoints included the 12-week changes from baseline in Brief Assessment of Cognition in Schizophrenia (BACS) score and Schizophrenia Cognition Rating Scale (SCoRS) score. Safety endpoints included adverse event assessments. The full analysis set included all randomized patients (N = 256 [placebo, n = 87; luvadaxistat 50 mg, n = 58; 125 mg, n = 56; 500 mg, n = 55]); 228 patients completed the study. No significant improvements in PANSS NSFS were observed at any dose versus placebo at week 12. Improvements were observed with luvadaxistat 50 mg versus placebo in cognitive endpoints: BACS composite score (nominal one-sided p = 0.031) and SCoRS interviewer total score (nominal one-sided p = 0.011). Luvadaxistat did not significantly improve negative symptoms of schizophrenia. However, luvadaxistat 50 mg met the prespecified secondary endpoints for cognitive performance (BACS) and function (SCoRS), warranting further investigation in patients with cognitive impairment associated with schizophrenia. Luvadaxistat was well-tolerated in INTERACT, with no new safety signals observed. ClinicalTrials.gov: NCT03382639.

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.

Improving the enzymatic activity and stability of N-carbamoyl hydrolase using deep learning approach.

BACKGROUND: Optically active D-amino acids are widely used as intermediates in the synthesis of antibiotics, insecticides, and peptide hormones. Currently, the two-enzyme cascade reaction is the most efficient way to produce D-amino acids using enzymes DHdt and DCase, but DCase is susceptible to heat inactivation. Here, to enhance the enzymatic activity and thermal stability of DCase, a rational design software "Feitian" was developed based on kcat prediction using the deep learning approach. RESULTS: According to empirical design and prediction of "Feitian" software, six single-point mutants with high kcat value were selected and successfully constructed by site-directed mutagenesis. Out of six, three mutants (Q4C, T212S, and A302C) showed higher enzymatic activity than the wild-type. Furthermore, the combined triple-point mutant DCase-M3 (Q4C/T212S/A302C) exhibited a 4.25-fold increase in activity (29.77 ± 4.52 U) and a 2.25-fold increase in thermal stability as compared to the wild-type, respectively. Through the whole-cell reaction, the high titer of D-HPG (2.57 ± 0.43 mM) was produced by the mutant Q4C/T212S/A302C, which was about 2.04-fold of the wild-type. Molecular dynamics simulation results showed that DCase-M3 significantly enhances the rigidity of the catalytic site and thus increases the activity of DCase-M3. CONCLUSIONS: In this study, an efficient rational design software "Feitian" was successfully developed with a prediction accuracy of about 50% in enzymatic activity. A triple-point mutant DCase-M3 (Q4C/T212S/A302C) with enhanced enzymatic activity and thermostability was successfully obtained, which could be applied to the development of a fully enzymatic process for the industrial production of D-HPG.

Effects of eyestalk ablation and seawater temperature on d-glutamate levels in the reproductive tissues of male kuruma prawn Marsupenaeus japonicus.

D-Glutamate, a novel d-amino acid found in animal tissues, exclusively exists in the male reproductive tissues of the kuruma prawn, Marsupenaeus japonicus. Herein, changes in the d-glutamate content were determined in the male reproductive tissues of M. japonicus during acclimation to breeding seawater temperatures of 18-22°C and unilateral eyestalk ablation. The d-glutamate content in the testis increased with increasing seawater temperature and with unilateral eyestalk ablation. This suggests that both stimulations induced d-glutamate synthesis in the testis. Although the d-alanine content in the testis increased after unilateral eyestalk ablation, it did not change with elevated seawater temperature. Furthermore, we determined the d-glutamate distribution in the M. japonicus spermatophore. This indicates that d-glutamate is crucial in prawn fertilization.

In vivo Labeling and Intravital Imaging of Bacterial Infection using a Near-infrared Fluorescent D-Amino Acid Probe.

Bacterial infections still pose a severe threat to public health, necessitating novel tools for real-time analysis of microbial behaviors in living organisms. While genetically engineered strains with fluorescent or luminescent reporters are commonly used in tracking bacteria, their in vivo uses are often limited. Here, we report a near-infrared fluorescent D-amino acid (FDAA) probe, Cy7ADA, for in situ labeling and intravital imaging of bacterial infections in mice. Cy7ADA probe effectively labels various bacteria in vitro and pathogenic Staphylococcus aureus in mice after intraperitoneal injection. Because of Cy7's high tissue penetration and the quick excretion of free probes via urine, real-time visualization of the pathogens in a liver abscess model via intravital confocal microscopy is achieved. The biodistributions, including their intracellular localization within Kupffer cells, are revealed. Monitoring bacterial responses to antibiotics also demonstrates Cy7ADA's capability to reflect the bacterial load dynamics within the host. Furthermore, Cy7ADA facilitates three-dimensional pathogen imaging in tissue-cleared liver samples, showcasing its potential for studying the biogeography of microbes in different organs. Integrating near-infrared FDAA probes with intravital microscopy holds promise for wide applications in studying bacterial infections in vivo.

FRBM Mini REVIEW: Chemogenetic approaches to probe redox dysregulation in heart failure.

Chemogenetics refers to experimental methods that use novel recombinant proteins that can be dynamically and uniquely regulated by specific biochemicals. Chemogenetic approaches allow the precise manipulation of cellular signaling to delineate the molecular pathways involved in both physiological and pathological disease states. Approaches utilizing yeast d-amino acid oxidase (DAAO) enable manipulation of intracellular redox metabolism through generation of hydrogen peroxide in the presence of d-amino acids and have led to the development of new and informative animal models to characterize the impact of oxidative stress in heart failure and neurodegeneration. These chemogenetic models, in which DAAO expression is regulated by different tissue-specific promoters, have led to a range of cardiac phenotypes. This review discusses chemogenetic approaches to manipulate oxidative stress in models of heart failure. These approaches provide new insights into the relationships between redox metabolism and normal and pathologic states in the heart, as well as in other diseases characterized by oxidative stress.

Multifunctionality of arginine residues in the active sites of non-canonical d-amino acid transaminases.

Structure-function relationships are key to understanding enzyme mechanisms, controlling enzyme activities, and designing biocatalysts. Here, we investigate the functions of arginine residues in the active sites of pyridoxal-5'-phosphate (PLP)-dependent non-canonical d-amino acid transaminases, focusing on the analysis of a transaminase from Haliscomenobacter hydrossis. Our results show that the tandem of arginine residues R28* and R90, which form the conserved R-[RK] motif in non-canonical d-amino acid transaminases, not only facilitates effective substrate binding but also regulates the catalytic properties of PLP. Non-covalent interactions between residues R28*, R90, and Y147 strengthen the hydrogen bond between Y147 and PLP, thereby maintaining the reactivity of the cofactor. Next, the R90 residue contributes to the stability of the holoenzyme. Finally, the R90I substitution induces structural changes that lead to substrate promiscuity, as evidenced by the effective binding of substrates with and without the α-carboxylate group. This study sheds light on the structural determinants of the activity of non-canonical d-amino acid transaminases. Understanding the structural basis of the active site plasticity in the non-canonical transaminase from H. hydrossis, which is characterized by effective conversion of d-amino acids and α-keto acids, may help to tailor it for industrial applications.

Anti-DNA antibody-targeted D-peptide nanoparticles ameliorate lupus nephritis in MRL/lpr mice.

Peptide ALW (ALWPPNLHAWVP) targeting anti-dsDNA antibodies has shown promising therapeutic effects in alleviating lupus nephritis, but is potentially limited by poor stability and non-kidney targeting. We recently developed a D-form modified ALW, called D-ALW, which has the capacity to widely inhibit pathogenic polyclonal anti-dsDNA antibody reactions. Further modification of D-ALW using PEG-PLGA nanoparticles to enhance good kidney-targeting ability and extend half-life. Here, we demonstrate that the D-form modified ALW maintains higher binding and inhibition efficiencies and achieves higher stability. Most importantly, D-ALW nanoparticles exhibit excellent kidney-targeting ability and prolong the half-life of the peptides in BALB/c mice. Additionally, compared to D-ALW, D-ALW nanoparticles significantly reduce the glomerular deposition of IgG and C3, improve renal histopathologies, such as glomerular proliferation and inflammatory cells infiltration, and markedly prolong lifespan in MRL/lpr lupus-prone mice. Overall, these results establish that the D-ALW nanoparticles offer synergistic benefits in both safety and efficacy, providing long-term renal preservation and treatment advantages in lupus nephritis.

Nontargeted Identification of D-Amino Acid-Containing Peptides Through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS.

D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.

Multifunctional enzymes related to amino acid metabolism in bacteria.

In bacteria, d-amino acids are primarily synthesized from l-amino acids by amino acid racemases, but some bacteria use d-amino acid aminotransferases to synthesize d-amino acids. d-Amino acids are peptidoglycan components in the cell wall involved in several physiological processes, such as bacterial growth, biofilm dispersal, and peptidoglycan metabolism. Therefore, their metabolism and physiological roles have attracted increasing attention. Recently, we identified novel bacterial d-amino acid metabolic pathways, which involve amino acid racemases, with broad substrate specificity, as well as multifunctional enzymes with d-amino acid-metabolizing activity. Here, I review these multifunctional enzymes and their related d- and l-amino acid metabolic pathways in Escherichia coli and the hyperthermophile Thermotoga maritima.

Biosynthesis and Degradation of Free D-Amino Acids and Their Physiological Roles in the Periphery and Endocrine Glands.

It was long believed that D-amino acids were either unnatural isomers or laboratory artifacts, and that the important functions of amino acids were exerted only by L-amino acids. However, recent investigations have revealed a variety of D-amino acids in mammals that play important roles in physiological functions, including free D-serine and D-aspartate that are crucial in the central nervous system. The functions of several D-amino acids in the periphery and endocrine glands are also receiving increasing attention. Here, we present an overview of recent advances in elucidating the physiological roles of D-amino acids, especially in the periphery and endocrine glands.

Investigating D-Amino Acid Oxidase Expression and Interaction Network Analyses in Pathways Associated With Cellular Stress: Implications in the Biology of Aging.

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids by oxidative deamination, producing hydrogen peroxide (H2O2) as a by-product. The generation of intracellular H2O2 may alter the redox-homeostasis mechanism of cells and increase the oxidative stress levels in tissues, associated with the pathogenesis of age-related diseases and organ decline. This study investigates the effect of DAO knockdown using clustered regularly interspaced short palindromic repeats (CRISPR) through an in silico approach on its protein-protein interactions (PPIs) and their potential roles in the process of aging. The target sequence and guide RNA of DAO were designed using the CCTop database, PPI analysis using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, Reactome biological pathway, protein docking using GalaxyTongDock database, and structure analysis. The translated target sequence of DAO lies between amino acids 43 to 50. The 10 proteins that were predicted to interact with DAO are involved in peroxisome pathways such as acyl-coenzyme A oxidase 1 (ACOX1), alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT), catalase (CAT), carnitine O-acetyltransferase (CRAT), glyceronephosphate O-acyltransferase (GNPAT), hydroxyacid oxidase 1 (HAO1), hydroxyacid oxidase 2 (HAO2), trans-L-3-hydroxyproline dehydratase (L3HYPDH), polyamine oxidase (PAOX), and pipecolic acid and sarcosine oxidase (PIPOX). In summary, DAO mutation would most likely reduce activity with its interacting proteins that generate H2O2. However, DAO mutation may result in peroxisomal disorders, and thus, alternative techniques should be considered for an in vivo approach.