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Amino acid racemases catalyze the interconversion of l- and d-amino acids, maintaining intracellular levels of both d- and l-amino acids. While alanine and glutamate racemases are widespread in bacteria, serine racemase (SerR) is predominantly found in animals. Recently, homologs of animal SerR were reported in some bacterial genomes, but their evolutionary distribution and functional roles remain poorly understood. In this study, we cloned and expressed 20 SerR homologous genes from 13 bacterial species spanning five phyla and characterized their enzymatic activity. Six homologs exhibited serine dehydratase activity, while the remaining showed racemase activity with serine, aspartate, asparagine, or arginine. Notably, the SerR homologs from Parafannyhessea umbonata (Actinomycetota), Clostridium aceticum, Anaerovirgula multivorans, Alkaliphilus oremlandii (Bacillota), Acetomicrobium mobile, and Thermovirga lienii (Synergistota) demonstrated strong arginine racemase activity, with Km values ranging from 0.167 to 0.885 mM and kcat values ranging from 5.86 to 61.5 s−1 for l-arginine. Phylogenetic analysis revealed that bacterial and eukaryotic SerR homologs share a common ancestral gene, and substrate specificity has independently changed multiple times during evolution. Amino acid sequence alignment and analysis of site-directed mutants revealed that residues at positions 146 to 148 and surrounding regions, located near the substrate-binding site, play a crucial role in substrate specificity and/or catalytic activity. These results highlight the evolutionary processes that drive functional diversification in serine racemase homologs. [ABSTRACT FROM AUTHOR]

The accumulation of D-amino acid-containing peptides is associated with age-related diseases such as Alzheimer's disease and cataracts, while glycosylation is an important modification of proteins and plays a key role in improving the physicochemical properties of peptides and facilitating their regulation in biological systems. This study investigates the effects of glycosylation position, glycan number, and monosaccharide structure on the conformation and enzymatic degradation of D-amino acid-containing peptides, using KYNEtWRSED (5-t) as a model peptide and six monosaccharides as model glycans. The results demonstrated that glycosylation inhibited the enzymatic degradation of 5-t in the presence of most serine-like proteases. However, in the presence of chymotrypsin, glycosylation with modified monosaccharides (except for β-D-GalNAc) promoted the degradation of 5-t. Furthermore, glycosylation had no effect on the cleavage site of 5-t. Molecular docking analysis revealed that the hydrogen bonding and electrostatic interactions between the glycopeptide and chymotrypsin were markedly strengthened, likely serving as a key determinant of the enzymatic effects. Collectively, these findings highlight the potential of glycosylation to enhance the therapeutic and biomedical applications of D-amino acid-containing peptides in disease treatment and drug design. [ABSTRACT FROM AUTHOR]

Background: Mast cells play an important role in allergic reactions by releasing potent mediators, including histamine and platelet-activating factor (PAF), upon activation. We recently reported that a biotinylated heptapeptide (peptide 2), D-Lys(Biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp, which is highly stable in the plasma, inhibits PAF bioactivity and anaphylactic hypothermia in vivo. However, the effects of this peptide on allergy induction by mast cells have not been investigated. Methods and Results: Several biotinylated heptapeptides, including peptide 2, inhibited IgE/antigen-stimulated degranulation in RBL-2H3 cells. Peptide 2 also markedly inhibited the degranulation induced by calcium ionophore A23187. All peptides (peptide 1, 2 and 3) inhibited activating stimuli-induced increase in intracellular Ca2+ levels. The transforming growth factor-alpha shedding assay indicated that peptide 2 antagonized against the histamine H1 receptor. Furthermore, peptide 2 inhibited histamine-induced rat paw edema. Conclusion: These results highlight the potential of peptide 2 in the prevention and treatment of various allergic diseases mediated by mast cell activation. [ABSTRACT FROM AUTHOR]

Abstract The relationship between D-AA metabolic enzymes and cancer development remains unclear. We aimed to investigate this relationship using mice deficient in D-AA-related metabolic enzymes. We examined mice lacking these enzymes for approximately 900 days and the effects of altered D-AA metabolism on cancer development based on lifespan, pathological findings, and gene expression. The lifespan of female DASPO -knockout (DASPO −/− ) mice was shorter than that of the other group mice; furthermore, these mice showed tumor-like masses in the liver, spleen, and small intestine. A pathological diagnosis of diffuse large B-cell lymphoma (DLBCL) was made. RNA sequencing of the liver samples showed specific alterations in the expression of 71 genes in DASPO −/− mice compared with that in wild-type B6 mice; RGS 1, MTSS1, and SMARCD 1 were identified as DLBCL-related genes. Patients with DLBCL exhibiting low DASPO expression demonstrated a shorter survival period than those showing high expression. However, the role of DASPO in DLBCL development is unclear. Therefore, future research should focus on B cells. DASPO may serve as novel biomarkers and therapeutic targets in cancer. Graphical abstract

The relationship between D-AA metabolic enzymes and cancer development remains unclear. We aimed to investigate this relationship using mice deficient in D-AA-related metabolic enzymes. We examined mice lacking these enzymes for approximately 900 days and the effects of altered D-AA metabolism on cancer development based on lifespan, pathological findings, and gene expression. The lifespan of female DASPO -knockout (DASPO−/−) mice was shorter than that of the other group mice; furthermore, these mice showed tumor-like masses in the liver, spleen, and small intestine. A pathological diagnosis of diffuse large B-cell lymphoma (DLBCL) was made. RNA sequencing of the liver samples showed specific alterations in the expression of 71 genes in DASPO−/− mice compared with that in wild-type B6 mice; RGS 1, MTSS1, and SMARCD 1 were identified as DLBCL-related genes. Patients with DLBCL exhibiting low DASPO expression demonstrated a shorter survival period than those showing high expression. However, the role of DASPO in DLBCL development is unclear. Therefore, future research should focus on B cells. DASPO may serve as novel biomarkers and therapeutic targets in cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The first monomeric pyridoxal-5′-phosphate (PLP)-dependent transaminase from a marine, aromatic-compound-degrading, sulfate-reducing bacterium Desulfobacula toluolica Tol2, has been studied using structural, kinetic, and spectral methods. The monomeric organization of the transaminase was confirmed by both gel filtration and crystallography. The PLP-dependent transaminase is of the fold type IV and deaminates D-alanine and (R)-phenylethylamine in half-reactions. The enzyme shows high stereoselectivity; no deamination of L-amino acids and (S)-phenylethylamine is detected. Structural analysis and subsequent mutagenesis led to the conclusion that the monomeric architecture of the enzyme is the only one possible and sufficient for stereoselectivity and PLP binding, but not for the overall double-substrate transamination reaction and the stability of the holo form with the reduced cofactor—pyridoxamine-5′-phosphate. These results extend the structural university of the PLP fold type IV enzymes and demonstrate the need for deeper analysis of the sequence–structure–function relationships in the transaminases. [ABSTRACT FROM AUTHOR]

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 k cat / K m 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 k cat / K m 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. • H154 was first experimentally identified to be the fatal residue for deamination. • H154 mutation boosted the amination towards pyruvic acid and bulky 2-keto acids. • Coupling H154 variants with W121L/H227I resulted in enhanced amination activities. • The first report on the contrary effects of H154 during deamination and amination. • A reminder to update the high-throughput screening for DAPDH amination activity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background and Aims Free D-amino acids, which have different functions from L-amino acids, have recently been discovered in various tissues. However, studies on the potential interactions between intestinal inflammation and D-amino acids are limited. We examined the inhibitory effects of D-alanine on the pathogenesis of intestinal inflammation. Methods We investigated serum D-amino acid levels in 40 patients with ulcerative colitis and 34 healthy volunteers. For 7 days [d], acute colitis was induced using dextran sulphate sodium in C57BL/6J mice. Plasma D-amino acid levels were quantified in mice with dextran sulphate sodium-induced colitis, and these animals were administered D-alanine via intraperitoneal injection. IFN-γ , IL-12p35 , IL-17A , and IL-23p19 mRNA expression in the colonic mucosa was measured using real-time polymerase chain reaction [PCR]. In vitro proliferation assays were performed to assess naïve CD4+ T cell activation under Th-skewing conditions. Bone marrow cells were stimulated with mouse macrophage-colony stimulating factor to generate mouse bone marrow-derived macrophages. Results Serum D-alanine levels were significantly lower in patients with ulcerative colitis than in healthy volunteers. Dextran sulphate sodium-treated mice had significantly lower plasma D-alanine levels than control mice. D-alanine-treated mice had significantly lower disease activity index than control mice. IFN-γ , IL-12p35 , IL-17A , and IL-23p19 mRNA expression levels were significantly lower in D-alanine-administered mice than in control mice. D-alanine suppressed naïve T cell differentiation into Th1 cells in vitro, and inhibited the production of IL-12p35 and IL-23p19 in bone marrow-derived macrophages. Conclusions Our results suggest that D-alanine prevents dextran sulphate sodium-induced colitis in mice and suppresses IL-12p35 and IL-23p19 production in macrophages. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic gram-negative pathogenic microorganism that poses a significant challenge in clinical treatment. Antibiotics exhibit limited efficacy against mature biofilm, culminating in an increase in the number of antibiotic-resistant strains. Therefore, novel strategies are essential to enhance the effectiveness of antibiotics against Pseudomonas aeruginosa biofilms. D-histidine has been previously identified as a prospective anti-biofilm agent. However, limited attention has been directed towards its impact on Pseudomonas aeruginosa. Therefore, this study was undertaken to explore the effect of D-histidine on Pseudomonas aeruginosa in vitro. Our results demonstrated that D-histidine downregulated the mRNA expression of virulence and quorum sensing (QS)-associated genes in Pseudomonas aeruginosa PAO1 without affecting bacterial growth. Swarming and swimming motility tests revealed that D-histidine significantly reduced the motility and pathogenicity of PAO1. Moreover, crystal violet staining and confocal laser scanning microscopy demonstrated that D-histidine inhibited biofilm formation and triggered the disassembly of mature biofilms. Notably, D-histidine increased the susceptibility of PAO1 to amikacin compared to that in the amikacin-alone group. These findings underscore the efficacy of D-histidine in combating Pseudomonas aeruginosa by reducing biofilm formation and increasing biofilm disassembly. Moreover, the combination of amikacin and D-histidine induced a synergistic effect against Pseudomonas aeruginosa biofilms, suggesting the potential utility of D-histidine as a preventive strategy against biofilm-associated infections caused by Pseudomonas aeruginosa. [ABSTRACT FROM AUTHOR]

Abstract MTT assay The chirality of amino acids affects their physiological functions. Recent studies uncovered potential physiological effects of D‐amino acids (D‐AAs) in nephropathy. Here, we explored the protective effects of exogenous D‐AAs on chronic kidney disease (CKD). First, by the 3‐(4,5‐dimethylthiazol‐2‐YI)‐2,5‐diphenyltetrazolium bromide (MTT) assay it was found that among the four D‐AAs studied (D‐glutamate (D‐Glu), D‐aspartic acid (D‐Asp) being the highest content in fermented yogurt, and D‐alanine (D‐Ala), D‐cysteine (D‐Cys), amino acids with renal protective potential), D‐Cys most significantly enhanced the viability of hypoxia‐induced injured HK‐2 cells, even better than its L‐analog, L‐Cys. Mitochondrial function analyzed by JC‐1 assay showed that 10 and 100 mM D‐Cys can significantly reduce the green/red fluorescence intensity by 16.1% (p

Abstract Since the identified standard genetic code contains 61 triplet codons of three bases for the 20 L-proteinogenic amino acids (AAs), no D-AA should be found in natural products. This is not what is observed in the living world. D-AAs are found in numerous natural compounds produced by bacteria, algae, fungi, or marine animals, and even vertebrates. A review of the literature indicated the existence of at least 132 peptide natural compounds in which D-AAs are an essential part of their structure. All compounds are listed, numbered and described herein. The two biosynthetic routes leading to the presence of D-AA in natural products are: non-ribosomal peptide synthesis (NRPS), and ribosomally synthesized and post-translationally modified peptide (RiPP) synthesis which are described. The methods used to identify the AA chirality within naturally occurring peptides are briefly discussed. The biological activity of an all-L synthetic peptide is most often completely different from that of the D-containing natural compounds. Analyzing the selected natural compounds showed that D-Ala, D-Val, D-Leu and D-Ser are the most commonly encountered D-AAs closely followed by the non-proteinogenic D-allo-Thr. D-Lys and D-Met were the least prevalent D-AAs in naturally occurring compounds. Graphical Abstract

In this study, a rapid, inexpensive, and accurate colorimetric sensor for detecting psychrophilic bacteria was designed, comprising gold (Au) nanoparticles (NPs) modified by d-amino acid (D-AA) as color-metric probes. Based on the aggregation of Au NPs induced by psychrophilic bacteria, a noticeable color shift occurred within 6 h. Depending on the various metabolic behaviors of bacteria to different D-AA, four primary psychrophilic bacteria in raw milk were successfully distinguished by learning the response patterns. Furthermore, the quantification of single bacteria and the practical application in milk samples could be realized. Notably, a rapid colorimetric method was constructed by combining Au/D-AA with antibiotics for the minimum inhibitory concentration of psychrophilic bacteria, which relied on differences in bacteria metabolic activity in response to diverse antibiotic treatments. Therefore, the method enables the rapid detection and susceptibility evaluation of psychrophilic bacteria, promoting clinical practicability and antibiotic management.

d‐Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l‐serine by serine racemase and degraded by d‐amino acid oxidase. In the present study, we identified a new d‐serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l‐serine and tetrahydrofolate (THF) to glycine and 5,10‐methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d‐serine dehydratase activity, which degrades d‐serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d‐serine dehydration and did not exhibit dehydratase activity toward l‐serine. Furthermore, SHMT did not use d‐serine as a substrate in the canonical hydroxymethyltransferase reaction. The d‐serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d‐serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat/Km) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d‐serine dehydratase activity of SHMT was physiologically important because d‐serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild‐type strain. Collectively, these results suggest that SHMT is involved not only in l‐ but also in d‐serine metabolism through the degradation of d‐serine. [ABSTRACT FROM AUTHOR]

Some aquatic invertebrates contain free d -alanine. We previously showed copious amounts of free d -glutamate, a novel d -amino acid, in the tissue of the male reproductive organs of Marsupenaeus japonicus. Herein, we clarified the distribution and potential role of d -glutamate and d -alanine in male reproductive tissues, namely the testis, vas deferens and seminal receptacle at different growth stages of M. japonicus. The percentage of d -glutamate to total glutamate was over 50% in these tissues. In particular, the content of d -glutamate was the most abundant in the vas deferens, the ratio of d -glutamate to total glutamate was approximately 80%. In contrast, d -alanine content was the lowest in the vas deferens among these tissues. d -Glutamate content was the highest when the prawn weighed 12 g, indicating that d -glutamate is actively synthesized in the younger stage. Our findings suggest that d -glutamate plays an important role in the reproductive functions of M. japonicus. [ABSTRACT FROM AUTHOR]

ABSTRACTProphages are prevalent in the marine bacterial genomes and reshape the physiology and metabolism of their hosts. However, whether and how prophages influence the microbial degradation of D-amino acids (D-AAs), which is one of the widely distributed recalcitrant dissolved organic matters (RDOMs) in the ocean, remain to be explored. In this study, we addressed this issue in a representative marine bacterium, Shewanella psychrophila WP2 (WP2), and its integrated prophage SP1. Notably, compared to the WP2 wild-type strain, the SP1 deletion mutant of WP2 (WP2ΔSP1) exhibited a significantly lower D-glutamate (D-Glu) consumption rate and longer lag phase when D-Glu was used as the sole nitrogen source. The subsequent transcriptome analysis identified 1,523 differentially expressed genes involved in diverse cellular processes, especially that multiple genes related to inorganic nitrogen metabolism were highly upregulated. In addition, the dynamic profiles of ammonium, nitrate, and nitrite were distinct between the culture media of WP2 and WP2ΔSP1. Finally, we provide evidence that SP1 conferred a competitive advantage to WP2 when D-Glu was used as the sole nitrogen source and SP1-like phages may be widely distributed in the global ocean. Taken together, these findings offer novel insight into the influences of prophages on host metabolism and RDOM cycling in marine environments.IMPORTANCEThis work represents the first exploration of the impact of prophages on the D-amino acid (D-AA) metabolism of deep-sea bacteria. By using S. psychrophila WP2 and its integrated prophage SP1 as a representative system, we found that SP1 can significantly increase the catabolism rate of WP2 to D-glutamate and produce higher concentrations of ammonium, resulting in faster growth and competitive advantages. Our findings not only deepen our understanding of the interaction between deep-sea prophages and hosts but also provide new insights into the ecological role of prophages in refractory dissolved organic matter and the nitrogen cycle in deep oceans.

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5′-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure–function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine—a reversible inhibitor and analogue of (R)-1-phenylethylamine—a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the β-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds. [ABSTRACT FROM AUTHOR]

Since the identified standard genetic code contains 61 triplet codons of three bases for the 20 L-proteinogenic amino acids (AAs), no D-AA should be found in natural products. This is not what is observed in the living world. D-AAs are found in numerous natural compounds produced by bacteria, algae, fungi, or marine animals, and even vertebrates. A review of the literature indicated the existence of at least 132 peptide natural compounds in which D-AAs are an essential part of their structure. All compounds are listed, numbered and described herein. The two biosynthetic routes leading to the presence of D-AA in natural products are: non-ribosomal peptide synthesis (NRPS), and ribosomally synthesized and post-translationally modified peptide (RiPP) synthesis which are described. The methods used to identify the AA chirality within naturally occurring peptides are briefly discussed. The biological activity of an all-L synthetic peptide is most often completely different from that of the D-containing natural compounds. Analyzing the selected natural compounds showed that D-Ala, D-Val, D-Leu and D-Ser are the most commonly encountered D-AAs closely followed by the non-proteinogenic D-allo-Thr. D-Lys and D-Met were the least prevalent D-AAs in naturally occurring compounds. [ABSTRACT FROM AUTHOR]

Grisemycin, salinipeptin, and cypemycin belong to the linaridin class of ribosomally synthesized and posttranslationally modified peptides that contain multiple dehydrobutyrine and D-amino acid residues. The biosynthetic gene clusters of these linaridins lack obvious candidate genes for the dehydratase and epimerase required to introduce dehydrobutyrine and D-amino acid residues, respectively. However, we previously demonstrated that the grisemycin (grm) cluster contained cryptic dehydratase and epimerase genes by heterologous expression of this biosynthetic gene cluster in Streptomyces lividans and proposed that two genes (grmH and grmL) with unknown functions catalyze dehydration and epimerization reactions. In this study, we confirmed that both GrmH and GrmL, which were shown to constitute a protein complex by a co-purification experiment, were required to catalyze the dehydration, epimerization, and proteolytic cleavage of a precursor peptide GrmA by in vivo experiments. Furthermore, we demonstrated that GrmH/GrmL complex accepted salinipeptin and cypemycin precursor peptides, which possess three additional amino acids. GrmH and GrmL in linaridin natural product biosynthesis constitute an enzyme complex catalyzing dehydration, epimerization, and leader peptide removal. [ABSTRACT FROM AUTHOR]

Optically pure D-amino acids are key chemicals with various applications. Although the production of specific D-amino acids has been achieved by chemical synthesis or with in vitro enzyme catalysts, it is challenging to convert a simple carbon source into D-amino acids with high efficiency. Here, we design an artificial metabolic pathway by engineering bacteria to heterologously express racemase and N- acetyltransferase to produce N- acetyl-D-amino acids from L-amino acids. This new platform allows the cytotoxicity of D-amino acids to be avoided. The universal potential of this acetylation protection strategy for effectively synthesizing optically pure D-amino acids is demonstrated by testing sixteen amino acid targets. Furthermore, we combine pathway optimization and metabolic engineering in Escherichia coli and achieve practically useful efficiency with four specific examples, including N- acetyl-D-valine, N- acetyl-D-serine, N- acetyl-D-phenylalanine and N- acetyl-D-phenylglycine, with titers reaching 5.65 g/L, 5.25 g/L, 8.025 g/L and 130 mg/L, respectively. This work opens up opportunities for synthesizing D-amino acids directly from simple carbon sources, avoiding costly and unsustainable conventional approaches. • A cost-effective and universal strategy was developed for optically pure D-amino acids. • In particular, a novel acetyl group protection strategy was employed to address the chemical properties of toxic D-amino acid. • Four kinds of N -acetyl-D-amino acids were successfully de novo synthesized by this platform. • This process provides a new biosynthetic platform for metabolic engineering. [ABSTRACT FROM AUTHOR]

The D-amino acid content of Ishizuchi-kurocha, a post-fermented tea produced in Ehime, Japan, was measured. Ishizuchi-kurocha mainly contains D-glutamic acid and D-alanine, but it also contains a small amount of D-aspartic acid. Two types of lactic acid bacteria, Lactiplantibacillus plantarum and Levilactobacillus brevis, are the main species involved in lactic acid fermentation during the tea fermentation process. Therefore, the D-amino acid-producing abilities of strains of these two species isolated from Ishizuchi-kurocha were examined. Specifically, the production of D-aspartic acid, D-alanine, and D-glutamic acid by L. brevis and L. plantarum strains was observed. The amount of D-aspartic acid produced by L. plantarum was low. D-glutamine was detected in culture supernatant but not in bacterial cells. D-arginine was detected in bacterial cells of the L. plantarum strains but not in the culture supernatant. Both the L. brevis and L. plantarum strains possessed at least three kinds of putative racemase genes: alanine racemase, glutamate racemase, and aspartate racemase. However, their expression and enzyme activity remain unknown. L. plantarum and L. brevis could play an important role in the production of D-amino acids in Ishizuchi-kurocha. In fact, Ishizuchi-kurocha is expected to possess the effective physiological activities of D-amino acids. [ABSTRACT FROM AUTHOR]

Background & Aims: D-amino acids, the chiral counterparts of protein L-amino acids, were primarily produced and utilized by microbes, including those in the human gut. However, little was known about how orally administered or microbe-derived D-amino acids affected the gut microbial community or gut disease progression. Methods: The ratio of D- to L-amino acids was analyzed in feces and blood from patients with ulcerative colitis (UC) and healthy controls. Also, composition of microbe was analyzed from patients with UC. Mice were treated with D-amino acid in dextran sulfate sodium colitis model and liver cholangitis model. Results: The ratio of D- to L-amino acids was lower in the feces of patients with UC than that of healthy controls. Supplementation of D-amino acids ameliorated UC-related experimental colitis and liver cholangitis by inhibiting growth of Proteobacteria. Addition of D-alanine, a major building block for bacterial cell wall formation, to culture medium inhibited expression of the ftsZ gene required for cell fission in the Proteobacteria Escherichia coli and Klebsiella pneumoniae, thereby inhibiting growth. Overexpression of ftsZ restored growth of E. coli even when D-alanine was present. We found that D-alanine not only inhibited invasion of pathological K. pneumoniae into the host via pore formation in intestinal epithelial cells but also inhibited growth of E. coli and generation of antibiotic-resistant strains. Conclusions: D-amino acids might have potential for use in novel therapeutic approaches targeting Proteobacteria-associated dysbiosis and antibiotic-resistant bacterial diseases by means of their effects on the intestinal microbiota community.

Over the past half-decade, scientists and clinicians have witnessed remarkable success of therapeutic peptides in both the diagnosis and the treatment of various diseases. Although peptide drugs have major advantages over other therapeutic modalities, poor physicochemical and proteolytic stability profiles are major setbacks that undercut their clinical application. An assortment of different chemical modifications coupled with suitable delivery technologies could potentially generate greatly improved peptide drugs that can be translated to the clinic. FDA approval of oral semaglutide (Rybelsus®) in 2019 for the management of type 2 diabetes marked a key milestone in the advancement of peptide therapeutics. Peptides have unique characteristics that make them highly desirable as therapeutic agents. The physicochemical and proteolytic stability profiles determine the therapeutic potential of peptides. Multiple strategies to enhance the therapeutic profile of peptides have emerged. They include chemical modifications, such as cyclization, substitution with d -amino acids, peptoid formation, N -methylation, and side-chain halogenation, and incorporation in delivery systems. There have been recent advances in approaches to discover peptides having these modifications to attain desirable therapeutic properties. We critically review these recent advancements in therapeutic peptide development. [ABSTRACT FROM AUTHOR]

Currently, there exists no accurate, noninvasive clinical imaging method to detect living bacteria in vivo. Our goal is to provide a positron emission tomography (PET) method to image infection by targeting bacteria-specific metabolism. Standard of care methodologies detect morphologic changes, image immunologic response to infection, or employ invasive tissue sampling with associated patient morbidity. These strategies, however, are not specific for living bacteria and are often inadequate to detect bacterial infection during fever workup. As such, there is an unmet clinical need to identify and validate new imaging tools suitable for noninvasive, in vivo (PET) imaging of living bacteria. We have shown that d-[methyl-11C]methionine (d-[11C]Met) can distinguish active bacterial infection from sterile inflammation in a murine infection model and is sensitive to both Gram-positive and Gram-negative bacteria. Here, we report an automated and >99% enantiomeric excess (ee) synthesis of d-[11C]Met from a linear d-homocysteine precursor, a significant improvement over the previously reported synthesis utilizing a d-homocysteine thiolactone hydrochloride precursor with approximately 75-85% ee. Furthermore, we took additional steps toward applying d-[11C]Met to infected patients. d-[11C]Met was subject to a panel of clinically relevant bacterial strains and demonstrated promising sensitivity to these pathogens. Finally, we performed radiation dosimetry in a normal murine cohort to set the stage for translation to humans in the near future.

Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) have been proposed as universal signaling molecules in plant stress responses. There are a growing number of studies suggesting that hydrogen sulfide (H2S) or Reactive Sulfur Species (RSS) are also involved in plant abiotic as well as biotic stress responses. However, it is still a matter of debate as to how plants utilize those RSS in their signaling cascades. Here, we demonstrate that d-cysteine is a novel candidate for bridging our gap in understanding. In the genus of the tiny water-floating fern Azolla, a rapid root abscission occurs in response to a wide variety of environmental stimuli as well as chemical inducers. We tested five H2S chemical donors, Na2S, GYY4137, 5a, 8l, and 8o, and found that 5a showed a significant abscission activity. Root abscission also occurred with the polysulfides Na2S2, Na2S3, and Na2S4. Rapid root abscission comparable to other known chemical inducers was observed in the presence of d-cysteine, whereas l-cysteine showed no effect. We suggest that d-cysteine is a physiologically relevant substrate to induce root abscission in the water fern Azolla.

d-Amino acids, rare enantiomers of amino acids, have been identified as biomarkers and therapeutic options for COVID-19. Methods for monitoring recovery are necessary for managing COVID-19. On the other hand, the presence of SARS-CoV2 virus in the blood is associated with worse outcomes. We investigated the potential of d-amino acids for assessing recovery from severe COVID-19. In patients with severe COVID-19 requiring artificial ventilation, the blood levels of d-amino acids, including d-alanine, d-proline, d-serine, and d-asparagine, which were lower than the normal range before treatment, quickly and transiently increased and surpassed the upper limit of the normal range. This increase preceded the recovery of respiratory function, as indicated by ventilation weaning. The increase in blood d-amino acid levels was associated with the disappearance of the virus in the blood, but not with inflammatory manifestations or blood cytokine levels. d-Amino acids are sensitive biomarkers that reflect the recovery of the clinical course and blood viral load. Dynamic changes in blood d-amino acid levels are key indicators of clinical course.

Various d‐amino acids have been found in a wide range of organisms, including mammals. Although the physiological functions of various d‐amino acids have been reported or suggested, the molecular basis of these biological functions has been elucidated in only a few cases. The identification of a d‐amino acid biosynthetic enzyme is a critical step in understanding the mechanism of the physiological functions of d‐amino acids. While in vivo functional screening can be a powerful tool for identifying novel metabolic enzymes, none of the existing organisms exhibit growth dependent on d‐amino acid other than d‐Ala and d‐Glu. Here, we report the first organism that exhibits non‐canonical d‐amino acid auxotrophy. We found that an Escherichia coli strain lacking the major d‐Ala and d‐Glu biosynthetic enzymes, alr, dadX, and murI, and expressing the mutated d‐amino acid transaminase (DAAT) gene from Bacillus sp. YM‐1 (MB3000/mdaat+) grew well when supplemented with certain d‐amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLP‐dependent enzymes of E. coli showed that MB3000/mdaat+ could detect weak and moonlighting racemase activity, such from cystathionine β‐lyase (MetC) and a negative regulator of MalT activity/cystathionine β‐lyase (MalY)—these exhibit only a few tenths to a few thousandths of the racemization activity of canonical amino acid racemases. We believe that this unique platform will contribute to further research in this field by identifying novel d‐amino acid‐metabolizing enzymes. [ABSTRACT FROM AUTHOR]

• Microbial communities in a shale gas platform from Weiyuan, Sichuan are identified. • d -amino acids enhance biocide mitigation of shale microbiome biocorrosion of X80. • A mixture of d -amino acids further reduce corrosion current density and weight loss. • Magnetite nanoparticles accelerate corrosion of X80 carbon steel. • The acceleration confirms extracellular electron transfer-MIC by the microbiome. Microbiologically influenced corrosion (MIC) in shale gas field is a major threat with the hydraulic fracturing fluid injected into the subsurface. In this study, the microbiome collected from a shale gas produced water sample was extracted and cultivated in ATCC 1249 medium modified with 10 g/L NaCl anaerobically at 30 °C. d -amino acids, which were reported as biocide enhancers, were found to enhance 2,2-dibromo-3-nitrilopropionamide (DBNPA) biocide on the mitigation of shale microbiome MIC on X80 carbon steel. The combination of 50 ppm (w/w) d -leucine + 50 ppm d -alanine + 1 ppm d -tyrosine had the best enhancement effect on 50 ppm DBNPA with 84 % less weight loss, and 67 % lower corrosion current density (i corr) compared to 50 ppm DBNPA alone. The corrosion data were consistent with the enhanced biofilm inhibition observation. The experimental data also indicated that d -tyrosine used alone at a low dosage of 1 ppm enhanced DBNPA considerably, with 44 % less weight loss and 47 % less i corr. The electrochemical results showed the positive response of shale gas microbiome biofilm to the injected magnetite nanoparticles indicating the extracellular electron transfer might be a main mechanism for its corrosion. [ABSTRACT FROM AUTHOR]

The yeast Vanrija (previously Cryptococcus) humicola strain UJ1 produces d-aspartate oxidase (DDO) only in the presence of d-aspartate in culture media. This article provides RNA-sequencing data to identify the differentially expressed genes (DEGs) in the yeast cells grown between l- and d-aspartate. RNA samples were prepared from the yeast cells grown in a culture medium containing 30 mM d-aspartate or l-aspartate as the sole carbon source and subjected to RNA sequencing on Illumina NovaSeq6000 platform. The clean reads obtained by removing adaptor sequences and low-quality reads from raw reads were submitted to the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJDB13570. The clean reads were subjected to differential gene expression analysis using DEGSeq to provide data on the upregulated and downregulated DEGs in the cells grown on d-aspartate. The DEGs were subjected to gene ontology (GO) and KEGG pathway enrichment analyses using GOSeq and KOBAS, respectively, to provide data on the possible biological functions of the DEGs. The data set obtained in this project might be helpful for further investigation of the effects of d-aspartate on cellular processes in yeast cells and other eukaryotic organisms.

Ribosomal incorporation of D-a-amino acids (DAA) and N-methyl-L-α-amino acids (MeAA) with negatively charged sidechains, such as D-Asp, D-Glu, MeAsp and MeGlu, into nascent peptides is far more inefficient compared to those with neutral or positively charged ones. This is because of low binding affinity of their aminoacyl-transfer RNA (tRNA) to elongation factor-thermo unstable (EF-Tu), a translation factor responsible for accommodation of aminoacyl-tRNA onto ribosome. It is well known that EF-Tu binds to two parts of aminoacyl-tRNA, the amino acid moiety and the T-stem; however, the amino acid binding pocket of EF-Tu bearing Glu and Asp causes electric repulsion against the negatively charged amino acid charged on tRNA. To circumvent this issue, here we adopted two strategies: (i) use of an EF-Tu variant, called EF-Sep, in which the Glu216 and Asp217 residues in EF-Tu are substituted with Asn216 and Gly217, respectively; and (ii) reinforcement of the T-stem affinity using an artificially developed chimeric tRNA, tRNAPro1E2, whose T-stem is derived from Escherichia coli tRNAGlu that has high affinity to EF-Tu. Consequently, we could successfully enhance the incorporation efficiencies of D-Asp, D-Glu, MeAsp and MeGlu and demonstrated for the first time, to our knowledge, ribosomal synthesis of macrocyclic peptides containing multiple D-Asp or MeAsp. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'. [ABSTRACT FROM AUTHOR]

SARS-CoV-2, one of the human RNA viruses, is widely studied around the world. Significant efforts have been made to understand its molecular mechanisms of action and how it interacts with epithelial cells and the human microbiome since it has also been observed in gut microbiome bacteria. Many studies emphasize the importance of surface immunity and also that the mucosal system is critical in the interaction of the pathogen with the cells of the oral, nasal, pharyngeal, and intestinal epithelium. Recent studies have shown how bacteria in the human gut microbiome produce toxins capable of altering the classical mechanisms of interaction of viruses with surface cells. This paper presents a simple approach to highlight the initial behavior of a novel pathogen, SARS-CoV-2, on the human microbiome. The immunofluorescence microscopy technique can be combined with spectral counting performed at mass spectrometry of viral peptides in bacterial cultures, along with identification of the presence of D-amino acids within viral peptides in bacterial cultures and in patients' blood. This approach makes it possible to establish the possible expression or increase of viral RNA viruses in general and SARS-CoV-2, as discussed in this study, and to determine whether or not the microbiome is involved in the pathogenetic mechanisms of the viruses. This novel combined approach can provide information more rapidly, avoiding the biases of virological diagnosis and identifying whether a virus can interact with, bind to, and infect bacteria and epithelial cells. Understanding whether some viruses have bacteriophagic behavior allows vaccine therapies to be focused either toward certain toxins produced by bacteria in the microbiome or toward finding inert or symbiotic viral mutations with the human microbiome. This new knowledge opens a scenario on a possible future vaccine: the probiotics vaccine, engineered with the right resistance to viruses that attach to both the epithelium human surface and gut microbiome bacteria. [ABSTRACT FROM AUTHOR]

Various lactic acid bacteria synthesize d-branched-chain amino acids (d-BCAA) during growth, but their physiological function remains largely elusive. The pyridoxal phosphate-dependent enzyme isoleucine 2-epimerase (ILEP) has been identified as the key enzyme responsible for d-BCAA biosynthesis. Comparative genomic analyses revealed that genes encoding ILEP and an uncharacterized amino acid-polyamine-organocation (APC) family transporter are adjacent in several d-BCAA-producing bacteria, suggesting a functional link between these two proteins in d-BCAA metabolism. In this study, we investigated the function of the APC family transporter from Lactobacillus fermentum (LfAAP). Using heterologous expression systems in Escherichia coli and Lactococcus lactis, we demonstrated that LfAAP functions as a non-stereospecific BCAA importer. Mutational analysis revealed that Ala119 and Met331 play critical roles in substrate recognition. Heterologous expression of LfAAP and/or LfILEP in a L. lactis strain, which lacks the ILEP-AAP genes operon, revealed that ILEP functions as both synthetic and catabolic enzyme for d-BCAA. Our findings suggest that the ILEP-AAP system contribute to storage and subsequent utilization of BCAA in a form that is less accessible by other organisms, providing a potential competitive advantage in microbial environments., (© 2025 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

The peptidoglycan stem peptides of the hyperthermophile Thermotoga maritima contain an unusual D-lysine (D-Lys) alongside the usual D-alanine and D-glutamate. We identified a Lys racemase that catalyzes racemization between L-Lys and D-Lys, and a diaminopimelate (Dpm) epimerase that catalyzes epimerization between LL-Dpm and meso-Dpm. Herein, we characterized a Dpm decarboxylase (TM1517) that catalyzes the conversion of meso-Dpm to L-Lys. TM1517 displayed high decarboxylase activity toward meso-Dpm but no activity toward LL-Dpm. D-Lys was not detected in the decarboxylation of meso-Dpm. The pH and temperature dependencies and kinetic parameters of decarboxylase activity were determined. Although other amino acid metabolizing activities of TM1517 were investigated, TM1517 did not exhibit any activities. Therefore, TM1517 is a Dpm decarboxylase associated with L- and D-Lys biosynthesis in T. maritima., (© The Author(s) 2025. Published by Oxford University Press on behalf of FEMS.)

Various D-amino acids play important physiological roles in mammals, but the pathways of their production remain unknown except for d-serine, which is generated by serine racemase. Previously, we found that Escherichia coli cystathionine β-lyase possesses amino acid racemase activity in addition to β-lyase activity. In the present work, we evaluated the enzymatic activities of human cystathionine γ-lyase, which shares a relatively high amino acid sequence identity with cystathionine β-lyase. The enzyme did not show racemase activity toward various amino acids including alanine and lyase and dehydratase activities were highest toward L-cystathionine and L-homoserine, respectively. The enzyme also showed weak activity toward L-cysteine and L-serine but no activity toward D-amino acids. Intriguingly, the pH and temperature profiles of lyase activity were distinct from those of dehydratase activity. Catalytic efficiency was higher for lyase activity than for dehydratase activity. [ABSTRACT FROM AUTHOR]

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated. [ABSTRACT FROM AUTHOR]

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d‐lysine alongside the usual d‐alanine and d‐glutamate. We previously identified a lysine racemase involved in d‐lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d‐alanine and d‐glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d‐amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d‐amino acids, but did not display activity towards l‐amino acids. It displayed high specific activities towards d‐homoserine and d‐glutamine as amino donors. The most preferred acceptor was 2‐oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat/Km) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)‐deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831‐integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild‐type strain. These results suggest that TM0831 is involved in d‐glutamate production. TM0831 is a novel d‐amino acid aminotransferase with racemase activity that is involved in the production of d‐amino acids in T. maritima. [ABSTRACT FROM AUTHOR]

Platelet‐activating factor (PAF) is an important lipid mediator of anaphylaxis and therefore can be an anti‐anaphylactic agent target. Recently, we reported that several synthetic biotinylated peptides containing a Tyr‐Lys‐Asp‐Gly sequence markedly inhibited the bioactivities of PAF in vitro and in vivo; it also inhibited anaphylactic reactions such as hypothermia, hypotension, and vascular permeability in vivo. Here, we report the anti‐anaphylactic effects of three biotinylated heptapeptides (peptide 1: H‐Lys(biotinyl)‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp‐OH, peptide 2: H‐D‐Lys(biotinyl)‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp‐OH, and peptide 3: H‐D‐Lys(biotinyl)‐Trp‐Tyr‐Lys‐Asp‐Gly‐D‐Asp‐OH). The experiment using tryptophan fluorescence spectroscopy showed that the interaction of peptides 2 and 3 with PAF was larger than that of peptide 1. Experiments using a rat model of hind paw edema showed that peptides 1, 3, and 2 inhibited PAF‐induced edema by 67.9%, 69.3%, and 79.3%, respectively. In a mouse model of anaphylaxis, both peptides 2 and 3 showed inhibitory effects on anaphylactic hypothermia, whereas peptide 1 did not. Furthermore, experiments involving in vitro rat plasma stability of peptides showed that both peptides 3 and 2 were more stable in plasma compared to peptide 1 (84.0%, 51.8%, and 0%, remained after 6 h, respectively). Our results suggest that both peptides 2 and 3 may show systemic and local inhibitory effects as anti‐anaphylactic agents targeting PAF. [ABSTRACT FROM AUTHOR]

d -amino acids have been actively examined since improved analytical techniques revealed their presence in animal bodies. Although D-Asp was identified in mammals earlier than D-Ser, research on D-Asp has lagged behind that on D-Ser, mainly because the target protein of D-Asp remains unknown. To date, the only reported functions of D-Asp are its roles in reproduction and suggested neuromodulatory functions. Since d -amino acids are also present in food, it is important to clarify their effects on gastrointestinal epithelial cells, which are always contacted after ingestion. Therefore, the present study examined the effects of d -amino acids on gastrointestinal tract basal cells. The effects of 11 types of amino acids (Ala, Arg, Asn, Asp, Gln, Glu, Leu, Lys, Pro, Ser, and Val) on the proliferation of three types of gastrointestinal epithelial cells (HGC-27, IEC-6, and Caco-2) were assessed. Although the proliferation of HGC-27 and Caco-2 was not affected by any of the 11 types of L- and d -amino acids, D-Asp inhibited the proliferation of IEC-6, derived from small intestinal epithelial cells, in concentration- and exposure time-dependent manners. The present study also examined uptake transporters, metabolic enzymes, and insulin signaling pathways; however, the mechanisms underlying the inhibitory effects of D-Asp on the proliferation of IEC-6 were not elucidated. A more detailed understanding of these mechanisms may lead to the development of pharmaceuticals as main drugs or formulation materials. Further studies are warranted on the physiological effects of d -amino acids, including D-Asp. • D-Asp inhibits intestinal epithelial cell proliferation only. • The inhibitory effect of D-Asp on cell proliferation is counteracted by the coexistence of L-Asp. • D-Asp has potential to be a novel target for a main drug or a raw material for drug formulation. [ABSTRACT FROM AUTHOR]

The sensitivity of currently available screening tools for urothelial carcinoma (UC) remains unsatisfactory particularly at early stages. Hence, we aimed to establish a novel blood-based screening tool for urothelial carcinoma. We measured serum d -amino acid levels in 108 and 192 patients with and without UC individuals in the derivation cohort, and 15 and 25 patients with and without UC in the validation cohort. Serum d -asparagine levels were significantly higher in patients with UC than in those without UC (p < 0.0001). We developed a novel screening equation for the diagnosis of urothelial carcinoma using d -asparagine in serum and estimated the glomerular filtration rate (eGFR). Serum d -asparagine levels adjusted for eGFR exhibited high performance in the diagnosis of UC (AUC-ROC, 0.869; sensitivity, 80.6 %; specificity, 82.7 %), even in early-stage UC (AUC-ROC: 0.859, sensitivity: 83.3 %, specificity: 82.3 %), which were previously misdiagnosed via urinary occult blood or urine cytology. This established strategy combined with urinary occult blood, improves diagnostic ability (sensitivity: 93.7 %, specificity: 70.1 %). • A novel screening equation is created to diagnose urothelial carcinoma (UC) using d -amino acids. • D-asparagine adjusted for eGFR is a potential tool for early detection of UC. • The new method combined with urinary occult blood testing improved diagnostic ability. [ABSTRACT FROM AUTHOR]

The pupae of lepidopterans contain high concentrations of endogenous d -serine. In the silkworm Bombyx mori , d -serine is negligible during the larval stage but increases markedly during the pupal stage, reaching 50% of the total free serine. However, the physiological function of d -serine and the enzyme responsible for its production is unknown. Herein, we identified a new type of pyridoxal 5′-phosphate (PLP)-dependent serine racemase (SR) that catalyses the racemization of l -serine to d -serine in B. mori. This silkworm SR (BmSR) has an N-terminal PLP-binding domain that is homologous to mammalian SR and a C-terminal putative ligand-binding regulatory-like domain (ACT-like domain) that is absent in mammalian SR. Similar to mammalian SRs, BmSR catalyses the racemization and dehydration of both serine isomers. However, BmSR is different from mammalian SRs as evidenced by its insensitivity to Mg2+/Ca2+ and Mg-ATP—which are required for activation of mammalian SRs—and high d -serine dehydration activity. At the pupal stage, the SR activity was predominantly detected in the fat body, which was consistent with the timing and localization of BmSR expression. The results are an important first step in elucidating the physiological significance of d -serine in lepidopterans. [ABSTRACT FROM AUTHOR]

Genetic code reprogramming has enabled us to ribosomally incorporate various nonproteinogenic amino acids (npAAs) into peptides in vitro. The repertoire of usable npAAs has been expanded to include not only l-α-amino acids with noncanonical sidechains but also those with noncanonical backbones. Despite successful single incorporation of npAAs, multiple and consecutive incorporations often suffer from low efficiency or are even unsuccessful. To overcome this stumbling block, engineering approaches have been used to modify ribosomes, EF-Tu, and tRNAs. Here, we provide an overview of these in vitro methods that are aimed at optimal expansion of the npAA repertoire and their applications for the development of de novo bioactive peptides containing various npAAs. [ABSTRACT FROM AUTHOR]

Surface coatings are designed to mitigate pervasive biofouling herald, a new era of surface protection in complex biological environments. However, existing strategies are plagued by persistent and recurrent biofilm attachment, despite the use of bactericidal agents. Herein, a chiral metal-organic framework (MOF)-based coating with conformal microstructures to enable a new anti-biofouling mode that involves spontaneous biofilm disassembly followed by bacterial eradication is developed. A facile and universal metal-polyphenol network (MPN) is designed to robustly anchor the MOF nanoarmor of biocidal Cu 2+ ions and anti-biofilm d-amino acid ligands to a variety of substrates across different material categories and surface topologies. Incorporating a diverse array of chiral amino acids endows the resultant coatings with widespread signals for biofilm dispersal, facilitating copper-catalyzed chemodynamic reactions and inherent mechano-bactericidal activities. This synergistic mechanism yields unprecedented anti-biofouling efficacy elucidated by RNA-sequencing transcriptomics analysis, enhancing broad-spectrum antibacterial activities, preventing biofilm formation, and destroying mature biofilms. Additionally, the chelation-directed amorphous/crystalline coatings can activate photoluminescent properties to inhibit the settlement of microalgae biofilms. This study provides a distinctive perspective on chirality-enhanced antimicrobial behaviors and pioneers a rational pathway toward developing next-generation anti-biofouling coatings for diverse applications., (© 2024 Wiley‐VCH GmbH.)