Newsletter 04/2025 edited by Loredano Pollegioni

Do you have news concerning the D-amino acids field to announce? Is there a relevant published paper to mention? Write us and take the advantage of this bimonthly Newsletter.

The Editor’s pick selection of the most intriguing papers is highlighted in yellow.

The SerMET project explores how L-serine biosynthesis is regulated in the brain by focusing on the “serinosome,” a metabolic complex formed by the three enzymes of the phosphorylated pathway. By investigating the mechanisms controlling the formation of this complex and its role in L-serine synthesis, the project aims to shed light on the regulation of D- serine and glycine homeostasis—crucial modulators of NMDA receptor function.

SerMET is funded by Fondazione Cariplo and led by Dr. Valentina Rabattoni under the mentorship of Prof. Loredano Pollegioni.

More  info:  www.theproteinfactory2.it/human-serinosome-sermet

D-AAs AND PATHOLOGIES:

A Novel Polymorphic Form of Sodium Benzoate (Ω-NaBen): Improved Solubility, Stability, Central Nervous System Effects, and Antipsychotic Activities via D- Amino Acid Regulation

Chang WH, Lai YA, Tsai HC, Mao YW, Tsai M, Kuo CS, Shih YY, Lu LP, Tan P, Tsai GE

CNS Neurol Disord Drug Targets. 2025 Jul 15. doi: 10.2174/0118715273359634250626102333.

The D-amino acid oxidase inhibitor sodium benzoate (NaBen) is known to possess antipsychotic and cognition- enhancing effects in animal models. Here, a novel crystalline polymorph of NaBen (abbreviated as Ω-NaBen) was developed. Ω-NaBen showed improved CNS exposure, resulted in higher levels of D-serine or/and D- alanine in the brain, and in MK-801-treated mice displayed enhanced effects in alleviating hyperactivity and stronger potency in relieving cognitive impairment. It also improved efficacy in relieving social deficit, a negative symptom model of schizophrenia.

Sodium benzoate, a D-amino acid oxidase inhibitor, improved short-term memory in patients with mild cognitive impairment in a randomized, double- blind, placebo-controlled clinical trial

Lin CH, Wang SH, Lane HY

Psychiatry Clin Neurosci. 2025 May 23. doi: 10.1111/pcn.13841.

Sodium benzoate (a D-amino acid oxidase inhibitor) has been reported to improve cognitive function in patients with mild Alzheimer disease; however, its efficacy for mild cognitive impairment (MCI, especially its core feature, impaired short-term memory) remains uncertain. Benzoate and placebo were well tolerated and benzoate therapy produced no additional side effect. Compared with placebo, sodium benzoate therapy, displayed a trend in improving overall cognitive function and significantly improved short-term memory. The authors concluded that larger studies are warranted to confirm the preliminary finding. 

Unravelling the role of L- and D- alanine in prostate cancer: a Positron Emission Tomography study in a genetic mouse model

Castellnou P, Gómez-Martínez M, Gómez-Vallejo V, Baz Z, López-Gallego F, Rondon- Lorefice I, Zabala-Letona A, Poot AJ, Mendizabal I, Carracedo A, Rejc, Llop J

Nucl Med Biol. 2025 Jun 30;148-149:109048. doi: 10.1016/j.nucmedbio.2025.109048.

This study investigated the biodistribution and tumour accumulation of D- and L-[methyl-¹¹C]alanine using positron emission tomography (PET) imaging in a genetic mouse model of prostate cancer. demonstrating that the D-enantiomer exhibited faster blood clearance, higher age-dependent kidney retention, and greater prostate lesion uptake at 7 months compared to the L-enantiomer. Histological analysis confirmed malignant lesions in the prostate of PTEN knockout mice, corroborating the PET imaging findings. 

Biological and Analytical Perspectives on D-Amino Acids in Cancer Diagnosis and Therapy

Uifălean A, Iacobescu M, Salanță LC, Hegheş SC, Moldovan RC, Iuga CA

Pharmaceuticals (Basel). 2025; 18(5):705. doi: 10.3390/ph18050705.

This review focuses on the role of D-amino acids in cancer, highlighting how the detection of altered levels of D-amino acids can be helpful in early detection, progression, or response to treatment in several malignancies, such as gastric, hepatic, colorectal, or breast cancer. 

Airway microbiota associated D-phenylalanine promotes non-small cell lung cancer metastasis through epithelial mesenchymal transition

Gao L, Liao H, Chen Y, Ye C, Huang L, Xu M, Du J, Zhang J, Huang D, Cai S, Dong H.

Journal of Translational Medicine. 2025); 23 (1), art. no. 673, doi: 10.1186/s12967-025-06701-1

Previous studies have reported that microbiota and metabolites significantly differ between healthy individuals and lung cancer patients. Bronchoalveolar lavage fluid (BALF) samples were collected from 30 non-small cell lung cancer (NSCLC) patients. Integrated pathogenic metagenomic and liquid chromatography-mass spectrometry (LC‒MS) analyses were employed. The omics data show that D-phenylalanine was notably upregulated in patients with metastasis and was positively related to Metamycoplasma salivarium. Intranasal administration of D-Phe promoted tumor intrapulmonary metastasis and induced epithelial mesenchymal transition (EMT) process in NSCLC mouse models. Moreover, D-Phe promotes the proliferation of non-small cell lung cancer cells and facilitates their migration and invasion via EMT. 

Theoretical Analysis of D-Serine and D-Asparagine as Biomarkers for Glomerular Filtration Rate

Tanaka R, Kakuta Y, Nonomura N, Kimura T

Chem Biodivers. 2025 Jun 29:e00579. doi: 10.1002/cbdv.202500579

This work reports on D-serine and D-asparagine as novel endogenous biomarkers for glomerular filtration rate (GFR) in chronic kidney disease (CKD). Their clearance strongly correlates with inulin clearance, demonstrating high reliability and reduced muscle mass dependency compared to creatinine-based methods. These results suggest that D-amino acids offer a promising, less invasive alternative for precise renal function assessment. 

Determination of urine volume and glomerular filtration rate using D-serine and D-asparagine

Tanaka R, Sakai S, Taniguchi A, Kawamura M, Higa-Maegawa Y, Matsumura S, Fukae S, Nakazawa S, Kimura-Ohba S, Horio M, Takahara S, Imamura R, Nonomura N, Mizui M, Isaka Y,

Kakuta Y, Kimura T

(2025) Nephrology Dialysis Transplantation, 40 (7), pp. 1374 – 1383, doi: 10.1093/ndt/gfae279

Measurement of glomerular filtration rate (GFR) is subject to inaccurate urine collection. This observational study included 209 living kidney transplant donors and recipients for whom GFR was measured using the clearance of inulin. The authors reported that D-Ser and D-Asn were nearly completely excreted in urine after glomerular filtration, enabling the estimation of urine volume and correct mGFR. Besides reflecting GFR, D- serine and D-Asn can be used to estimate urine volume: by using the estimated urine volume (eUV) method, mGFR determined using clearance methods becomes more accurate.

Serum metabolomics reveals systemic metabolic alterations of Graves’ disease before and after antithyroid drug treatment

Li T, Li L, Wang X, Li Y, Gong Y, Zhou H, Zheng X.

Endocr Connect. 2025; 14(6):e250078. doi: 10.1530/EC-25-0078

Untargeted metabolomics was used to analyze changes of serum metabolites before and after 1 year methimazole treatment in patients with Graves’ disease (GD). Pathway enrichment analysis indicated significant alterations in tyrosine metabolism, biosynthesis of alkaloids derived from histidine and purine, and bile secretion pathways in untreated GD patients. In addition, pathways such as ABC transporters, folate biosynthesis, D- amino acid metabolism, anthranilate degradation, and purine metabolism remained significantly dysregulated after treatment. 

Synergistic behavioral and neuroplastic effects of psilocybin-NMDAR modulator administration

Ben-Tal T, Pogodin I, Botvinnik A, Lifschytz T, Heresco-Levy U, Lerer B

Translational Psychiatry. 2025; 15 (1), art. no. 200, doi: 10.1038/s41398-025-03428-x

The therapeutic potential of serotonergic psychedelics (SP) in treating neuropsychiatric disorders (depression and schizophrenia) is limited by possible adverse effects. In this work, the synergistic benefits of combining psilocybin (PSIL) with N-methyl-D-aspartate receptor (NMDAR) modulators D-serine (D-Ser) and D-cycloserine (D-CS) was investigated. The results on ICR male mice indicate that PSIL significantly increased HTR—a measure for hallucinogenic effects—which was reduced by the co-administration of D-Ser or D-CS in a dose-dependent manner. Similarly, combining PSIL with D-Ser or D-CS significantly decreased MK-801-induced hyperactivity, modelling antipsychotic effects. The PSIL-D-Ser combination enhanced GAP43 expression and overall expression of the 4 assayed synaptic proteins in the hippocampus, while PSIL-D-CS elevated PSD95 levels, suggesting a synaptogenic synergy. These studies support the hypothesis that combinations of SP with NMDAR modulators could optimize the therapeutic potential of SP by mitigating adverse effects and enhancing neuroplasticity.

Early involvement of D-serine in β-amyloid-dependent pathophysiology

Billard J.-M., Ploux E., Largilliere S., Corvaisier S., Gorisse-Hussonnois L., Radzishevsky I., Wolosker H., Freret T.

Cellular and Molecular Life Sciences. 2025; 82 (1), art. no. 179, doi: 10.1007/s00018-025- 05691-z

The beta-amyloid (Aß) peptide which accumulates in Alzheimer’s disease (AD), affects the D-serine-dependent NMDA receptos activation in vitro, but whether this alteration would significantly contribute to AD-related pathophysiology and memory deficits remains unclear. This study report on a decrease in the maximal pool of recruitable NMDA receptors and in the expression of NMDA receptor-dependent long-term potentiation together with impaired basal neurotransmission at CA3/CA1 synapses from hippocampal slices of 5xFAD mouse, an AD- related model with elevated Aß levels. The NMDA-related synaptic deregulations (but not the altered basal neurotransmission) and behavioral impairments are prevented or reduced in 5xFAD mice devoid of D-Ser after genetic deletion of its synthesis enzyme serine racemase. This evidence links D-Ser at least in the early pathogenic signatures of AD driven by the increase in Aß load and casts doubts on the recent proposal of preventive therapy of AD by administration of the precursor L-Ser.

D-Serine disrupts Cbln1 and GluD1 interaction and affects Cbln1-dependent
synaptic effects and nocifensive responses in the central amygdala

Sabnis SS, S Narasimhan KK, Chettiar PB, Shelkar GP, Dravid SM

Cellular and Molecular Life ciences. 2025; 82 (1), art. no. 67, doi: 10.1007/s00018-024- 05554-z

This study reports that D-serine inhibited the interaction between Cbln1 and GluD1 in an in vitro cell-binding assay. Furthermore, in ex vivo central amygdala (CeA) slices application of recombinant Cbln1 (rCbln1) produced a robust increase in excitatory neurotransmission and GluD1 expression, an effect partially blocked by pre- treatment with D-Ser. Furthermore, the pro-nociceptive effect of intra-CeA injection of rCbln1 was inhibited by D-Ser pre-treatment and the antinociceptive effect of intra-CeA rCbln1 injection in an inflammatory pain model was blocked by D-Ser. This evidence demonstrated that D-Ser binding to GluD1 reduces its interaction with Cbln1, which may be relevant to synaptic plasticity and behaviour. 

Impacts of D-aspartate on the Aggregation Kinetics and Structural Polymorphism of Amyloid β Peptide 1–42

Hsiao L-C, Lee C-H, Mazmanian K, Yoshida M, Ito G, Murata T, Utsunomiya-Tate N, Haino T, Tate S-I, Hsu S-TD

Journal of Molecular Biology. 2025; 437 (12), art. no. 169092, doi: 10.1016/j.jmb.2025.169092

This work reports on the isomerization of L-Asp at different positions of amyloid β peptide 1–42 (Aβ42). The structures of Aβ42 amyloid fibrils harbouring a single D-Asp at position 23 and two D-Asp at positions 7 and 23 were solved by cryo-electron microscopy helical reconstruction, revealing how D-Asp7 contributes to the formation of a unique triple stranded amyloid fibril structure stabilized by two threads of well-ordered water molecules. These findings provide crucial insights into how the conversion from L- to D-Asp influences the age- dependent aggregation propensity and amyloid polymorphism of Aβ42. 

Bisphenol A in utero induced glutamate and D-serine metabolic dysregulation in the hippocampus of rats and primary cultured astrocytes

Zhang L., Li X., Zhao Y., Wang P., Shi M., Li X., Pei X., Duan Z., Ma M., Yu H.

Ecotoxicology and Environmental Safety. 2025; 302, art. no. 118651, doi: 10.1016/j.ecoenv.2025.118651

Bisphenol A (BPA) is a widely used synthetic compound that could cause neurobehavioral abnormalities in mammals. Previous studies have suggested that NMDAR may be a potential target of BPA-induced neurotoxicity. Pregnant SD rats were exposed at increasing BPA amounts via oral gavage from gestational day (GD) 5 to GD 19, as well as primary cultured astrocytes (AS) from neonatal rats. BPA exposure in utero induced:

1) Glu accumulation and inhibited GS, GLS1, and GDH expression and activity in the hippocampus of rats’ offspring at different developmental stages (GD 20, PND 21, and PND 56), 2) increased D-Ser levels at GD 20 while decreased them from PND 21 onward, 3) inhibited SR, asc-1, and ASCT2 expression, while promoting ASCT1 expression during these stages, d) up-regulated DAAO expression at GD 20 but down-regulated it from PND 21 onward. Moreover, BPA exposure inhibited the expression and activity of GS, GLS1, and GDH, while suppressing SR and DAAO expression but increasing ASCT2 expression without altering ASCT21 expression in AS.

D-AAS & PHYSIOLOGICAL ROLES:

D-Alanine content in the marine edible bivalve Panopea  japonica and evaluation of its associated enzyme activities

Onozato M, Takaura T, Shinohara W, Tsukada T, Sakamoto T, Okoshi K, Fukushima T Sci Rep. 2025 Jul 14;15(1):25415. doi: 10.1038/s41598-025-10379-2

This study investigated the amino acid composition of the geoduck clam Panopea japonica, emphasizing its remarkably high D-alanine (D-Ala) content in the siphon tissue: 6.99-14.2 mmol/100 g-wet amounted to 91-94% of the total Ala, far exceeding that of other bivalves such as Tresus keenae (74%). Alanine racemase and D- amino acid oxidase activities are present suggesting active D-Ala biosynthesis and metabolism. The high concentrations of D-Ala enhance its value as a delicacy owing to its unique sweetness.

Kynurenic Acid Synthesis from D-Kynurenine in the Cerebellum: A Distinct Role
of D-Amino Acid Oxidase

Pérez de la Cruz V, Sathyasaikumar KV, Wang XD, Blanco Ayala T, Beggiato S, González Esquivel DF, Pineda B, Schwarcz R

Cells. 2025 Jul 5;14(13):1030. doi: 10.3390/cells14131030

The enzymatic formation of kynurenic acid (KYNA), a neuromodulator metabolite of the kynurenine pathway of tryptophan metabolism, in the mammalian brain is widely attributed to kynurenine aminotransferase II (KATII). However, an alternative biosynthetic route, involving the conversion of D-kynurenine (D-KYN) to KYNA by D-amino acid oxidase (DAAO), may play a role as well. This work confirmed that purified DAAO efficiently converted D- KYN-(but not L-KYN) into KYNA and showed the production of KYNA from D-KYN (100 µM) in vitro using tissue homogenates from several human brain regions: the most effective was the cerebellum. Rat cerebellum KYNA neosynthesis was significantly reduced by DAAO inhibition, whereas KATII inhibition had no effect. Finally, KYNA production was assessed by in vivo microdialysis in rat cerebellum. 

D-amino acid metabolic versatility as a common adaptive strategy in the Mariana Trench microbiome

Wang X, Lv Y, Zhao W, Xiao X, Wang J

mSystems.  2025  Jul  11:e0058125.  doi:  10.1128/msystems.00581-25

Deep-sea microorganisms play a crucial role in the turnover of recalcitrant dissolved organic matter (RDOM). A comprehensive reference database of D-AA functional genes was evaluated for accurate identification of D- AA metabolic potential from metagenomic data: various D-AA anabolic and catabolic genes that were closely correlated with central carbon metabolism and ammonia oxidation genes were identified throughout the water column and in the sediment of the Mariana Trench. E.g., glutamate racemase is ubiquitously present in ammonia-oxidizing archaea. The work also reported an increase in both D-AA production and degradation potential with water depth, with higher levels in near-bottom seawater than in sediment.

Regulation of Gene Expression of Mouse D-Amino Acid Oxidase Trinh HTT, Shishido Y, Tran NH, Tran DH, Kim SH, Sogabe H, Fukui K Chembiochem. 2025 Jun 18:e202500323. doi: 10.1002/cbic.202500323

Previous studies on human DAAO encoding gene identified two promoter regions (P1 and P2), a negative regulatory element in intron 1, and several transcription factor binding sites. In this study, the regulatory mechanism of mouse DAAO gene expression was compared with the human system. The highest promoter activity was detected in the -333/-87 subregion, with residual activities in the -87/+111 region. Bioinformatics analysis identified transcription factors, including NEUR, EGRF, ZF07, ZF11, KLFS, SP1F and ZF02, which bind to both the human and mouse DAAO genes at conserved positions. 

Mammalian Tolerance to Amino Acid Heterochirality

Taniguchi S, Adachi K, Tran X, Suzuki M, Sasabe J

Chembiochem. 2025 Jul 11;26(13):e202500273. doi: 10.1002/cbic.202500273

This manuscript reviews how the chiral balance of free D-amino acids or residues in proteins is maintained in mammals at the individual aThis manuscript reviews how the chiral balance of free D-amino acids or residues in proteins is maintained in mammals at the individual and cellular levels.nd cellular levels. 

Aspartate in the Brain: A Review

Rae C.D., Rowlands B.D., Balcar V.J.

(2025) Neurochemical Research, 50 (3), art. no. 199, doi: 10.1007/s11064-025-04454-3

L-aspartate plays key roles in metabolism as an amino donor and acceptor. It contributes to the synthesis of protein, arginine and nitric oxide, asparagine, N-acetylaspartate and N-methyl-D-aspartate. The role of D- aspartate in brain function is more likely to be involved in fine regulation of endocrine and homeostatic processes. Perturbations of aspartate metabolism have been described in a range of neurological deficits, particularly those of white matter. This review reports on the various roles of aspartate in the brain, its metabolism, transport and compartmentation, its role as a neurotransmitter or a more general signalling molecule, with specific focus on its role(s) in disease processes.

ENZYMES ACTIVE ON D-AAS:

Structural determinants of unique substrate specificity of D-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii

Shimekake Y, Furuichi T, Imanishi D, Takahashi S

Enzyme Microb Technol. 2025 Jul 1;190:110705. doi: 10.1016/j.enzmictec.2025.110705

D-Amino acid oxidase from the thermophilic fungus Rasamsonia emersonii (ReDAAO) shows high stability and broad substrate specificity. Comparing ReDAAO with TdDAAO from the thermophilic fungus Thermomyces dupontii, revealed that ReDAAO lacks the YVLQG loop present in TdDAAO, which exhibited narrower substrate specificity. Inserting the YVLQG loop into ReDAAO narrowed its substrate specificity to match TdDAAO, while deleting the sequence from TdDAAO broadened its substrate specificity, resembling ReDAAO. The activity of ReDAAO toward D-Glu appears to depend on Arg97 and Ser231: alanine scanning at these residues significantly reduced D-Glu activity.

Structural determinants of the thermostability of D-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii

Furuichi T, Shimekake Y, Imanishi D, Takahashi S

J Biosci Bioeng. 2025 Jun 27:S1389-1723(25)00137-9. doi: 10.1016/j.jbiosc.2025.06.003

In order to identify the rationale of the thermostability of ReDAAO, thermolabile variants of ReDAAO with a single amino acid substitution (L134P, K203E, C230S, V275G, and V305L), were generated. The thermostabilization conferred by the disulfide bond and the interaction network involving K203 were unique to thermophilic fungal DAAOs. 

Elucidation of multifunctionality and substrate specificity of human aspartate aminotransferases

Miyamoto T, Kito H, Sato K, Sugiki T, Sakai-Kato K

Biochim Biophys Acta Proteins Proteom. 2025, 30;1873(5):141081. doi:10.1016/j.bbapap.2025.141081

The substrate specificity of human aspartate aminotransferases (hGOT1 and hGOT2) was investigated to reveal whether they possess D-amino acid metabolic activity. Neither enzyme displayed racemase activity toward various amino acids including aspartate (only a slight alanine racemase activity was detected). Likewise, neither  exhibited lyase, dehydratase, or aspartate decarboxylase activities. Both enzymes displayed high aminotransferase activity toward L-aspartate and L-glutamate as amino donors (also acting on other L-amino acids), but not D-amino acids. The aminotransferase activity for oxaloacetate followed a sigmoidal kinetics. 

Suicide substrate reaction-like modification of mouse serine racemase with L- serine

Hata A., Ito T., Mori H., Ogawa T., Kurihara T., Hemmi H., Yoshimura T.

Journal of Biochemistry.2 2025; 177 (6), pp. 437 – 445, DOI: 10.1093/jb/mvaf019

This study reports that mouse SR (mSR, a pyridoxal 5′-phosphate-dependent fold-type II serine racemase) underwent a suicide substrate reaction-like modification with its substrate, resulting in a remarkable change in its reaction specificity. mSR gradually lost part of its activity by the incubation with L- and D-Ser. The active site lysine residue of mSR was modified with an α-aminoacrylate intermediate generated from L-Ser and converted to a lysinoalanine residue. The modification significantly decreased the racemization and L-Ser dehydration activities, while dramatically increased the D-Ser dehydration activity by strong decrease of the Km value, favouring D-Ser degradation by mSR under physiological conditions. 

Rosetta-Driven Rapid Evolution of meso-Diaminopimelate Dehydrogenases for Structurally Diverse D-Amino Acid Production

Qiu H, Gong W, Huang H, Guo H, Gao Z, Liu L, Yin X

J Agric Food Chem. 2025 Jun 25;73(25):15847-15859. doi: 10.1021/acs.jafc.5c03073

Here, a Rosetta-driven evolution approach was used to tune meso-diaminopimelate dehydrogenases (DAPDH) substrate specificity. The activities of two DAPDHs toward model substrates─ benzoylformic acid and 3- indolepyruvic acid were enhanced from undetectable levels to 29.5 and 5.1 U/mg, respectively, this allowing the synthesis of structurally diverse bulky D-amino acids at both analytical and preparative scales. 

Revisiting D-Acylases for D-Amino Acid Production

Martínez-Rodríguez S, Gavira JA

Microb Biotechnol. 2025 Jun;18(6):e70179. doi: 10.1111/1751-7915.70179

N-Acyl-D-amino acid deacylases are used in the kinetic resolution of N-acetyl-D,L-amino acids (NAAs) due to a marked stereospecificity, and used with an N-succinyl-amino acid racemase (NSAR) in the dynamic kinetic resolution of different NAAs until the corresponding enantiomerically pure D-amino acids. Two new recombinant D-acylases from Bordetella petrii and Klebsiella pneumoniae were characterized and coupled with the recombinant Geobacillus stearothermophilus NSAR for the biosynthesis of D-methionine or D-aminobutyric acid. Furthermore, the D-acylase from Klebsiella pneumoniae was also characterized, highlighting the importance of an α/β mobile domain in the substrate specificity. 

Directed Evolution of an (R)-Selective Transaminase Toward Higher Efficiency of Sitagliptin Analog Biosynthesis

Jia D.-X., Zang L., Ni C.-D., Wang J.-L., Yu H., Liu Z.-Q., Zheng Y.-G.

Biotechnology and Bioengineering. 2025; 122 (7), pp. 1735 – 1746, doi: 10.1002/bit.28988

Sitagliptin is an antihyperglycemic drug to treat type II diabetes. Herein, an efficient (R)-selective transaminase (TA) was used to synthesize the sitagliptin analog (R)-3-amino-1-morpholino-4-(2,4,5-trifluorophenyl)butan-1- one. Starting from (R)-ATA5 variant, two rounds of directed evolution combined with error-prone PCR, site- directed saturation and combinatorial mutagenesis yielded ATA5/F189H/S236T/M121H variant showing a 10-fold higher activity and a 4-fold improved half-life at 45 °C. This enzyme catalyzes the amination of 700 mM substrate with a conversion up to 93% and product e.e.> 99% in a cosolvent reaction system, and transforms 200 mM substrate with a conversion of 98% and product e.e. > 99% in a cosolvent-free system. 

Soluble and pocket engineering of D-amino acid oxidase and its application in the biotransformation of L-glufosinate

Kang X, Xu J, Wang Z, Yu S, Qin L, Zhang B, Zhou S, Yang L

Enzyme Microb Technol. 2025 May 24;190:110677. doi: 10.1016/j.enzmictec.2025.110677

L-glufosinate (L-PPT), a compound with high herbicidal activity, is produced from the kinetic resolution of commercially available D,L-PPT through four-enzyme one-pot biocatalysis. D-amino acid oxidase (DAAO) catalyzes the conversion of D-PPT to 2-oxo-4-[(hydroxy)(methyl)phosphinyl] butyric acid, playing a critical role in the synthesis of L-PPT. This study identified NcDAAO from Neurospora crassa OR74A, with increased solubility following the combination of N-terminal fusion tags with protein sequence truncation strategies, and 15-fold increase enzyme activity. The V117N/Q325S variant demonstrated enhanced catalytic activity toward the substrate D-PPT and was used for the conversion of D-PPT to L-PPT, achieving an enantiomeric excess > 99 %.

D-AAS & BIOTECHNOLOGY:

Widely Targeted Metabolomics Reveals Metabolic Divergence in Abutilon theophrasti Populations Under Glufosinate Ammonium Treatment

Guo X, Wang Y, Guo Y, Luo C, Cong K

Plants (Basel). 2025 Jun 30;14(13):1994. doi: 10.3390/plants14131994

Abutilon theophrasti Medikus, a pervasive weed infesting transgenic corn fields, exhibits increasing tolerance to the herbicide glufosinate ammonium. This study employed a widely targeted metabolomics approach to investigate differential metabolic responses to glufosinate ammonium. Three metabolic pathways (arginine and proline metabolism, biosynthesis of amino acids, D-amino acid metabolism) were identified as critical regulators of herbicide response.

An integrative approach for imaging and quantitative analysis of gut microbiota growth in vivo using fluorescent D-amino acid labeling and fluorescence in situ hybridization

Zhou Y, Lin L, Wang W

Biophys Rep. 2025 Jun 30;11(3):172-179. doi: 10.52601/bpr.2024.240044

Here, a protocol that integrates fluorescent D-amino acid (FDAA) metabolic labeling with fluorescence in situ hybridization (FISH) was used for imaging and analyzing the in vivo growth of gut microbiota. By administering two FDAAs sequentially through mouse gavage, the peptidoglycan of gut bacteria was labelled in their native environment, to serve as markers of cellular proliferation and division. The intensity of FDAA labelling directly correlates with the metabolic activity of gut bacteria. Additionally, FISH distinguished specific bacterial taxa of interest via fluorescence microscopy or flow cytometry.

Hazelnut: Explorations towards the biocatalytic synthesis of its aroma
precursor

Salvadó-Pau M, Fessner WD, Findrik Blažević Z, Breuer M

Chembiochem. 2025 May 22:e202500192. doi: 10.1002/cbic.202500192

In this work a novel biosynthetic route for the synthesis of filbertone (5-methyl-2-hepten-4-one, 1, which is the principal flavour compound of hazelnut) has been reported. This pathway consists of a multi-enzyme cascade, which starts from D-Ile and L-Thr that are individually converted by D-amino acid oxidase and threonine deaminase, respectively, followed by C-C ligation of the obtained products catalysed by a regioselective transketolase (TK) from Geobacillus stearothermophilus, allowing a potentially sustainable production of the natural aroma.

Protocol  for  measuring  D-amino  acid  transport  in  mammalian  cells  using  a fluorescent biosensor

Johal D, Hewton KG, Gale SJ, Johal AS, Parker SJ

STAR Protoc. 2025 Jun 28;6(3):103929. doi: 10.1016/j.xpro.2025.103929

This paper reports about a protocol to visualize amino acid transport in mammalian cells in real time using an enzyme-based biosensor.

Prolonged In Vivo Chemogenetic Generation of Hydrogen Peroxide by
Endothelial Cells Induces Cardiac Remodelling and Vascular Dysfunction

Lopez M, Herrle N, Amirmiran B, Malacarne PF, Werkhäuser J, Chatterjee S, Kader C, Jurisch V, Wen X, Gheisari M, Schäfer K, Münch C, Leuschner F, Gilsbach R, Rezende F, Brandes RP Antioxidants (Basel). 2025 Jun 10;14(6):705. doi: 10.3390/antiox14060705

This work reports on an inducible, endothelial cell-specific knock-in mouse model expressing a yeast D-amino acid oxidase (DAAO). DAAO produces hydrogen peroxide during D-amino acids oxidation. The induction of these DAAO mice was performed with tamoxifen, then received D-alanine (0.5 M) through drinking water, and the effects on ROS production and vascular and cardiac function were determined. DAAO induction increased endothelial ROS production as well as ROS production in the lung, with limited functional consequences: The authors concluded that acute stimulation of endothelial ROS improves cardiovascular function, whereas prolonged ROS exposure deteriorates it. 

Cryo-EM structures of GnRHR: Foundations for next-generation therapeutics

Shen S, He X, Liu H, Hu W, Xu HE, Duan J

Proc Natl Acad Sci U S A. 2025 Jun 24;122(25):e2500112122. doi: 10.1073/pnas.2500112122

Gonadotropin-releasing hormone receptor (GnRHR) is critical for reproductive health and a key therapeutic target for endocrine disorders and hormone-responsive cancers. Here, the structure of Sus scrofa and Xenopus laevis GnRHRs bound to mammal GnRH were solved using high-resolution cryoelectron microscopy. Structure- activity relationship analysis demonstrates how D-amino acid substitutions in GnRH analogs enhance stability and receptor affinity, useful insights for designing next-generation GnRHR therapeutics. 

One-pot synthesis of fluorescent nanoprobes based on D-Cys-based CDs and quantitative detection of lysine enantiomers

Li J, Wang F, Zhang H, Cao D, Guan R

Spectrochimica Acta – Part A: Molecular and Biomolecular Spectroscopy, 339, art. no. 126143. doi: 10.1016/j.saa.2025.126143

Chiral carbon dots (CDs) have attracted the attention of researchers. Here, a fluorescence probe (CYS-CDs + Cu²⁺) was constructed using D-Cys as chiral source to synthesize chiral carbon dots (CYS-CDs), and Cu²⁺ as inducer to recognize the chirality of lysine enantiomers. Cu²⁺ shows the ability to quench the fluorescence of CYS-CDs and L-lysine, but not D-Lys, could restore the fluorescence of the CYS-CDs + Cu²⁺ probe, yielding an “on–off–on” mode to detect L-Lys. This probe can be used for the quantitative detection of L-Lys by  fluorescence method (0–520 μM detection range and 13.7 μM limit of detection). In addition, a paper-based fluorescent sensor was constructed.

Ornithine enantiomers modulate essential oil yield and constituents and gene expression of monoterpenes synthase in Salvia officinalis under well-watered and drought stress conditions

Mohammadi-Cheraghabadi M., Ghanati F., Karimi N., Ghorbanpour M., Hazrati S. BMC Plant Biology. 2025; 25 (1), art. no. 148. doi: 10.1186/s12870-025-06156-y

This study examined the effects of L- and D-ornithine (1 mM) under both well-watered and drought stress conditions on the growth traits, essential oil (EO) yield, and composition, gene expression, and total phenolic and flavonoid content of Salvia officinalis. The drought stress led to a decrease in plant biomass and an increase in EO content, chemical profiles of the EO, and total phenolic and flavonoid content. However, the exogenous supplementation of ornithine, particularly D-ornithine, resulted in enhanced stem, leaf, and total plant biomass, a 20% increase in EO content, and a 75% increase in yield. Additionally, these were increases of 12% in total phenol and 70%, 106%, and 114% in flavonoid content when compared to well-watered plants without ornithine supplementation. D-ornithine enhanced the expression of BS and SS by 45% and 114%, respectively, under drought stress, while both D-ornithine and D,L-ornithine significantly increased CS expression. 

D-Alanine functionalized Iridium(III) complexes as two-photon photo-antibiotics for bacteria-specific ablation in infected macrophages

Shu J., Jiang H., Lin M., Liang J., Zhao Y., Luo D., Wang J., Chao H.

European Journal of Medicinal Chemistry. 2025; 294, art. no. 117758, DOI: 10.1016/j.ejmech.2025.117758

A novel photodynamic antibacterial therapeutic strategy was generated by functionalizing D-alanine on Iridium(III) complexes. The synergistic D-Ala metabolic labelling function and two-photon photodynamic eradication capacity of Ir(III) complexes enable bacterial imaging and elimination of bacterial pathogens within host cells. These effectively inhibit bacterial biofilm formation and efficiently eliminate intracellular bacterial infections in macrophages, enabling real-time dynamic monitoring of antimicrobial efficacy.

D-AAS IN BACTERIA:

D-Histidine inhibits Streptococcus mutans growth as a potential anti-caries agent

Yang L, Chen Y, Chi Y, Chen X, Zhao Y, Zhang M, Wang X, Li Y, Nie J, Wang X

J Oral Microbiol. 2025 Jul 16;17(1):2533174. doi: 10.1080/20002297.2025.2533174

This study demonstrated that D-His significantly inhibits the planktonic growth of S. mutans and delays biofilm formation, particularly in the early stages. RNA sequencing revealed significant alterations in pathways related to carbohydrate utilization, protein biosynthesis, and transmembrane transport following D-His-treatment. D-His exhibited effective caries prevention in an in vivo rat model.

D-Amino acids affect Pseudomonas aeruginosa biofilm and quorum sensing molecules in lung infection models developed under a cystic fibrosis environment

Rosado-Rosa JM, Parmar D, Rubakhin SS, Shrout JD, Sweedler JV Sci Rep. 2025 Jul 13;15(1):25328. doi: 10.1038/s41598-025-10519-8

This study explored the effects of six individual D-amino acids (alanine, aspartic acid, tyrosine, glutamic acid, serine, and proline) on the quorum sensing signalling and biofilm biomass production of two strains: PAO1 and the CF isolate FRD1. D-Asp (and D-Pro) caused the most significant decrease in biofilm mass and a decrease in quorum sensing molecule, while D-glutamic acid and D-serine had the opposite effects. 

D-alanine synthesis and exogenous alanine affect the antimicrobial susceptibility of Staphylococcus aureus

Suzuki Y, Kawada-Matsuo M, Thuan VTT, Le MN-T, Sakaguchi T, Komatsuzawa H Antimicrob Agents Chemother. 2025 Jul 2;69(7):e0193624. doi: 10.1128/aac.01936-24

D-alanine (D-Ala) is important for peptidoglycan biosynthesis in Staphylococcus aureus and is used for the modification of teichoic acids to weaken the net surface negative charge, leading to decreased susceptibility to cationic antimicrobial agents. D-Ala synthesis is dependent on alanine racemase (alr1 gene) and D-amino acid transaminase (dat gene), while the uptake of L- and D-Ala is dependent on the alanine transporter CycA. The alr1, dat, and cycA inactivation mutants presented antimicrobial increased susceptibility to β-lactams, D- cycloserine, bacitracin, lysostaphin, and cationic antimicrobial agents. Furthermore, in an Ala-depleted medium, the MIC for oxacillin decreased significantly, and the MIC for gentamicin also decreased slightly. Clinical MRSA strains also showed significantly increased susceptibility to oxacillin in the Ala-depleted medium. These results indicate that D-Ala deficiency leads to impaired peptidoglycan and increased net surface negative charge, resulting in increased antimicrobial susceptibility. 

Phenotypic plasticity in cell elongation among closely related bacterial species Delaby  M,  Yang  L,  Jacq  M,  Gallagher  KA,  Kysela  DT,  Hughes  V,  Pulido  F,  Veyrier  FJ, VanNieuwenhze MS, Brun YV

Nat Commun. 2025 Jun 2;16(1):5099. doi: 10.1038/s41467-025-60005-y

This work uses fluorescent D-amino acids to track the spatiotemporal dynamics of bacterial cell elongation, revealing unsuspected diversity of elongation modes among closely related species of the family Caulobacteraceae. Multiple cell elongation modes in bacteria have been described, even because their underlying mechanisms are targets of numerous antibiotics.

D-AAS PEPTIDES AND PROTEINS:

Potency of all-D amino acid antimicrobial peptides derived from the bovine rumen microbiome on tuberculous and non-tuberculous mycobacteria

Boidin-Wichlacz C, Maresca M, Correia I, Lequin O, Point V, Casanova M, Reinbold A, Iranzo O, Huws SA, Brodin P, Oyama LB, Tasiemski A, Canaan S, Cavalier JF

Curr Res Microb Sci. 2025 Apr 22;8:100395. doi: 10.1016/j.crmicr.2025.100395.

Mycobacterium tuberculosis is the world’s leading cause of death from a single infectious agent, thus antimicrobial peptides (AMPs) derived from the bovine rumen microbiome have shown promise against many resistant pathogens. The Lynronne 1, 2 & 3 and P15s AMPs from bovine rumen microbiome, as well as their all-D amino acid enantiomers, were evaluated against non-tuberculous and tuberculous mycobacteria, as well as their respective cytotoxicity against human cell lines and hemolytic activity on human erythrocytes. Although all- D enantiomers showed increased cytotoxicity to human cell lines, they still offer a good therapeutic window with improved activity compared to their L-form counterparts, especially Lynronne 2D all and P15sD. These peptides mostly act by insertion into the mycobacterial membrane resulting in a rapid membranolytic effect. 

Salt-Tolerant, Protease-Stable and Non-Resistance Developing Cationic AMPs  for Combatting Planktonic MRSA and its Biofilms

Yadav M, Singh T, Singh HV, Thummer RP, Chatterjee S

ACS Med Chem Lett. 2025 May 5;16(6):1089-1097. doi: 10.1021/acsmedchemlett.5c00121.

This study developed highly potent, salt-tolerant, nontoxic, and proteolytically stable membranolytic AMPs: D- WRL (composed of all D-amino acids) and W-(Dab)-L (incorporating 2,4-diaminobutyric acid), which effectively prevented planktonic MRSA, inhibited biofilm formation, and eradicated  80% of mature biofilms, outperforming vancomycin with faster killing kinetics. The biofilm eradication ability was attributed to their protease stability. The developed AMPs prevented resistance development in MRSA over 96 generations. 

De novo design of D-peptide ligands: Application to influenza virus hemagglutinin

Juraszek J., Kadam R.U., Branduardi D., van Ameijde J., Garg D., Dailly N., Jongeneelen M., Vermond J., Brakenhoff J.P.J., Brandenburg B., van Dongen M.J.P., Vogels R., Friesen R.H.E., Wilson I.A.

Proceedings of the National Academy of Sciences of the United States of America. 2025; 122 (26), art. no. e2426554122, doi: 10.1073/pnas.2426554122

This study reports on a computational method for de novo design of D-peptides that bind to an epitope of interest on the target protein using Rosetta’s hotspot-centric approach. The approach enables more facile design of D-peptides and its applicability is demonstrated by design of D-peptidic binders of influenza A virus hemagglutinin, as confirmed by 3D studies.

Modulating Peptide Self-Assembly via Triblock Chiral Patterning O’Neill CL, Fascetti JL, Clapacs Z, Kaplita LK, Liu CY, Kim D, White MA, Rudra JS Chemistry. 2025 Jul 2;31(37):e202404603. doi: 10.1002/chem.202404603

This work reports on a combinatorial exploration of all triblock chiral patterns for the model β-sheet-forming peptide KFE12 (Ac-(FKFE)3-NH2), revealing that each produces a unique morphology, ranging from minimal 4- nm-wide fibrils to micron-scale semi-structured aggregates. This study illustrates a combination of conserved and divergent hierarchical features, reflecting complex interplay between persistent fundamental forces and the unique spatial implications of blockwise intramolecular chiral interfaces.

D-AAS AND ANALYTICAL METHODS:

D-Amino acid analysis in solution using the photochemical properties of protonated adenosines

Nakanishi R, Fujihara A

Photochem Photobiol Sci. 2025 Jul;24(7):1153-1158. doi: 10.1007/s43630-025-00751-6

The chiral-recognition ability of protonated adenosine was investigated using a tandem mass spectrometer equipped with an electrospray ionization source and cold ion trap. The ultraviolet photodissociation spectra of the hydrogen-bonded protonated clusters of adenosine and histidine enantiomers at 100 K revealed that H+ (adenosine)(D-His) exhibited a 2-fold higher relative intensity at 278 nm than that of H+(adenosine)(L-His). His enantiomers were identified by probing the electronic excited state of the adenine moiety in protonated adenosine in the gas phase. The adenine moiety acquires the ability to recognize chirality via its glycosidic bond with D-ribose in protonated adenosine, that was used in quantitative-analysis applications in solution. 

Multicolumn Two-Dimensional Liquid Chromatography Screening Platform for Stereopeptidomics and Application to Antimicrobial Peptide Polyene and Lipopeptide

Knappe C, Jaag SJ, Dema T, Jaufmann R, Buckenmaier S, Gross H, Grond S, Lämmerhofer M Anal Chem. 2025 Jul 8;97(26):14048-14057. doi: 10.1021/acs.analchem.5c02658

This paper reports on a multicolumn two-dimensional liquid chromatography-tandem high-resolution mass spectrometry (2D LC-HRMS) platform with multiple reversed-phase type columns in the first dimension (1D) and multiple chiral columns in the second dimension (2D) for the determination of stereointegrity and absolute configurations of therapeutic peptides.

Enabling enantioselective and chemoselective fluorescence discrimination of D- threonine with concurrent stereochemical profiling in feed matrices

Yu X., Zhang B., Fan C., Liu Q., Sun P., Liu Y., Dong Q., Shu W., Chi W., Zeng C. Microchemical Journal. 2025; 215, art. no. 114514, doi: 10.1016/j.microc.2025.114514

This study reports on the synthesis of a pair of bifunctional chiral fluorescent probes, (R)-D1 and (S)-D1, utilizing binaphthol as the chiral fluorescence scaffold with recognition groups positioned at the 2,2′ sites for the selective recognition of chiral threonine (Thr), critical for ensuring food quality, safeguarding human and poultry health. The probe (R)-D1 demonstrates enantioselectivity and chemoselectivity in recognizing D-Thr, while (S)-D1 exhibits a complementary recognition for L-Thr. These probes show a broad detectable concentration range (0– 500 equivalents of amino acids) and were successfully applied to detect chiral Thr in feed samples. 

Chiral ligand-exchange capillary electrochromatography with thermo- responsive poly(N,N-dimethylacrylamide) as coating for efficient separation of D,L-amino acids

Ali N, Liu Y, Wang F, Qi L

Talanta. 2025; 293, art. no. 128090, doi: 10.1016/j.talanta.2025.128090

Herein, enantioseparation was performed by regulating hydrophobic/hydrophilic interactions in smart polymer through varying external environmental conditions: a chiral ligand exchange-capillary electrochromatography- UV system was built introducing a thermo-responsive poly(styrene-maleic anhydride-N,N-dimethylacrylamide) to the coating for enantioseparation of dansylated D,L-amino acids (Dns-D,L-AAs). A total of 15-pairs were separated at 40 °C, compared to 4-pairs baseline separated at 20 °C among 16-pairs of Dns-D,L-AAs. The proposed method showed an excellent linear dependence relation between concentration of L-alanine and UV absorbance intensity and good repeatability with relative standard deviations less than 1.9% for migration time and less than 3.9% for resolution.

INFORMATION

The D-amino acids International Research Center “DAAIR“ has been established in Gerenzano (Varese, Italy) in 2019 with the aim to support and perform scientific research projects and activities on the field of D-amino acids. The Center, located inside the Fondazione Istituto Insubrico Ricerca per la Vita, is aimed to represent a pole of excellence at international level for dissemination and research involving the D-amino acids (Director Silvia Sacchi).

Info@d-aminoacids.com

director@d-aminoacids.com

D-AMINO ACIDS INTERNATIONAL RESEARCH CENTER

INFO@D-AMINOACIDS.COM