It is a pleasure to announce that the 6th International Conference of D-Amino Acid Research (IDAR2024) and 18th Joint Conference of the D-Amino Acid Research Society Japan will be held at Kanazawa University in August 2024 between 21 (Wednesday) and 24 (Saturday). 
The website of IDAR2024 is open now, please visit the following:

The key dates are as follows.
March 1, 2024    Call for abstract open
March 1, 2024    Online registration open
June 14, 2024     Deadline for abstract submission
June 30, 2024     Deadline for early bird registration





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


D-Amino acids: new clinical pathways for brain diseases.
Souza INO, Roychaudhuri R, de
Belleroche J, Mothet JP. Trends Mol Med. 2023 Dec;29(12):1014-1028. doi:10.1016/j.molmed.2023.09.001.
This interesting and seminal review aims to give an overview on the interlaced roles of three D-amino acids (D-AAs) that were recognized to form a new class of neuromodulators critical for higher cognitive functions, D-Ser, D-Asp, and D-Cys, in the healthy and diseased brain. Authors show how their metabolic pathways are dynamically regulated and cross other biochemical processes, discuss how disturbances in their metabolism cause developmental and homeostatic alterations, and illustrate that mutations in the genes encoding their metabolic enzymes confer high risk for major brain disorders. Finally, they outline promising directions for translating our current knowledge of D- AAs to the clinic as prognostics biomarkers and drug targets.

D-amino Acids Ameliorate Experimental Colitis and Cholangitis by Inhibiting Growth of
Proteobacteria: Potential Therapeutic Role in Inflammatory Bowel Disease.

Umeda S, Sujino T, Miyamoto K, Yoshimatsu Y, Harada Y, Nishiyama K, Aoto Y, Adachi K,Hayashi N, Amafuji K, Moritoki N, Shibata S, Sasaki N, Mita M, Tanemoto S, Ono K, Mikami Y,Sasabe J, Takabayashi K, Hosoe N, Suzuki T, Sato T, Atarashi K, Teratani T, Ogata H, NakamotoN, Shiomi D, Ashida H, Kanai T. Cell Mol Gastroenterol Hepatol. 2023;16(6):1011-1031. doi:

Patients with ulcerative colitis (UC) with an increased complement of Actinobacteria, Bacteroidetes, and Proteobacteria experience dysbiosis. In particular, several microorganisms are increased in inflammatory bowel disease (IBD)-related extraintestinal diseases. For this reason, antimicrobial therapy has been exploited to treat IBD and IBD-related diseases by regulating the numbers of Proteobacteria. However, the effectiveness of antibiotic therapy is limited owing to a reduction in beneficial bacteria and the emergence of antibiotic-resistant bacteria. Here, Umeda and collaborators elucidate the mechanism by which oral supplementation with D-amino acids (D-aa) suppress the growth and functional activity of Proteobacteria that show increased abundance in pUCs. The reported findings suggest that D-aa administration represents a potentially useful therapeutic approach for shaping the normal microbe community, not only for various dysbiosis-inducing diseases, but also for infections with antibiotic-resistant bacteria.


Cross species review of the physiological role of D-serine in translationally relevant behaviors.
Arizanovska D, Emodogo JA, Lally AP, Palavicino-Maggio CB, Liebl DJ, Folorunso OO. Amino
Acids. 2023 Oct 13. doi: 10.1007/s00726-023-03338-6.

D-serine is critical for complex behaviors, such as cognition and social behavior, where dysregulation of its synthesis and release has been implicated in many pathological conditions. This review, summarizes findings on the effect of D-serine on cognition, social interaction, and sleep. Moreover, it discusses D-serine in behaviors that are translationally relevant to multiple psychiatric and neurological disorders in different models across species (from fruit flies to humans), and highlights its potential as a therapeutic target.

Hydrogen sulfide and polysulfides induce GABA/glutamate/D-serine release, facilitate
hippocampal LTP, and regulate behavioral hyperactivity.
Furuie H, Kimura Y, Akaishi T, Yamada M, Miyasaka Y, Saitoh A, Shibuya N, Watanabe A,
Kusunose N, Mashimo T, Yoshikawa T, Yamada M, Abe K, Kimura H. Sci Rep. 2023 Oct
31;13(1):17663. doi: 10.1038/s41598-023-44877-y.

Here Furuie and coworkers, showed that Hydrogen sulfide (H2S) and polysulfides (H2Sn, n ≥ 2), small signaling molecules produced by 3‐mercaptopyruvate sulfurtransferase (3MST), induce the release of GABA, glutamate, glycine and D‐serine. Furthermore, they propose that H2S/H2Sn, 3MST, and the target of H2S2, transient receptor potential ankyrin 1 (TRPA1) channels, have therapeutic potential for psychiatric diseases and cognitive deficits, since studies in knocked-out mice revealed that their activation play a critical role in LTP induction, and the lack of 3MST causes behavioral hypersensitivity to NMDA receptor antagonism, as in schizophrenia.



Engineering acetylation platform for the total biosynthesis of D-amino acids.
Bi Y, Wang J, Li J, Chou HH, Ren T, Li J, Zhang K. Metab Eng. 2023 Nov;80:25-32.
doi: 10.1016/j.ymben.2023.09.001.


In this interesting piece of work, authors engineer E. coli to heterologously express a racemase anda N-acetyltransferase in an artificial metabolic pathway for converting L-amino acids into D-aminoacids and further protect them by acetylation. The new platform allows the cost-effective synthesisof optically pure various D-amino acids directly from simple carbon sources, avoiding cytotoxicitydue to their high intracellular concentration.

Rational design of the genetic code expansion toolkit for in vivo encoding of D-amino acids.
Jiang HK, Weng JH, Wang YH, Tsou JC, Chen PJ, Ko AA, Söll D, Tsai MD, Wang YS. Front
Genet. 2023 Oct 13;14:1277489. doi: 10.3389/fgene.2023.1277489.

D-amino acids (DAAs) have been used to generate optically active peptides and proteins for therapeutic purposes or novel protein design. However, in vivo incorporation of DAAs in proteins remains challenging and so far, largely limited due to endogenous translational machineries’ exclusive preference of L- over DAAs. To overcome this limitation, the catalytic pocket of the pyrrolysyl-tRNA synthetase (PylRS) derived from the archaeal Methanosarcina mazei has been extensively engineered: through amber suppression, the evolved enzyme is capable in incorporating various non-canonical amino acids. In this interesting paper, by generating new PylRS variants via rational design, the authors expand the scope of ribosomal protein synthesis, adding non-canonical DAAs (D-phenylalanine analogs) into the toolbox of protein engineering.

Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction
Kawamura Y, Ishida C, Miyata R, Miyata A, Hayashi S, Fujinami D, Ito S, Nakano S. Commun
Chem. 2023 Sep 22;6(1):200. doi: 10.1038/s42004-023-01005-1.

This work is the continuation of previous studies in which L-amino acid oxidases (LAAOs) exhibiting exceptional thermostability and long-term stability (being thus suitable for keto acid synthesis from L-amino acids) were designed by ancestral sequence reconstruction (ASR). Due to the limited substrate selectivity, low specific activity, and lack of 3D structures for the structure-based design of improved variants, the enzymatic application of these hyper-thermostable ancestral LAAOs (HTAncLAAOs) has been hurdled. Here, with the aim to optimize the enzymatic production of enantio-pure D-Trp derivatives (expected to be precursors of indole containing fine chemicals), Kawamura and co-workers design HTAncLAAO variants (HTAncLAAO2) by ASR to obtain the enzyme crystal structure and perform structure-guided mutagenesis of HTAncLAAO2 for improving the activity toward L-Trp. They claim that the obtained structural information will prove invaluable in designing variants with tailor-made substrate selectivity, and the generation of new oxidases with high activity towards various amines or amino acids.

Enhancement of the substrate specificity of D-amino acid oxidase based on tunnel-pocket
Wang L, Tang H, Zhu H, Xue Y, Zheng Y. Biotechnol Bioeng. 2023
Dec;120(12):3557-3569. doi: 10.1002/bit.28541.

In this study, authors generate an engineered variant of the D-amino acid oxidase of Rhodotorula taiwanensis (RtwDAAO) to be applied to the production of the L-phosphinothricin (L‐PPT) herbicide, a glutamate analog, by the stereoselective oxidation of the inactive enantiomer D‐PPT that is subsequently reduced to obtain optically pure L‐PPT. The low enzymatic activity of DAAO towards D‐PPT poses a major obstacle for industrial applications, therefore multiple highly active mutants of DAAO have been obtained by performing iterative site‐saturation mutations (ISM) at different sites, selected as mutation hotspots. Three rounds of ISM were performed based on the theoretical sequence of substrates passing through the enzyme tunnel and then into the active site pocket. Strikingly, the optimal produced variant M3rd‐SHVG exhibits a >2000‐fold increase in relative activity on the unnatural substrate D‐PPT.





Targeted Mutation of a Non-catalytic Gating Residue Increases the Rate of Pseudomonas
aeruginosa D-Arginine Dehydrogenase Catalytic Turnover.

Quaye JA, Ouedraogo D, Gadda G. J Agric Food Chem. 2023 Nov 7;71(45):17343–52. doi:


Commercial methods to synthesize L-amino acids rely on engineered D-amino acid oxidizing enzymes for detecting D-enantiomers and resolve racemic amino acid mixtures. However, there is still a limited understanding of enzyme catalysis, which is crucial for developing effective and improved biocatalysts. In this work, Quaye and co-workers engineer the flavin-dependent enzyme Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH), a good candidate for application in the L-amino acid and food industries since it oxidizes most D-amino acids to ketoacids and ammonia. They used site-directed mutagenesis, steady-state, and rapid reaction kinetics to generate enzyme variants substituted in Glu246 (a loop residue close to the active site, interacting with the substrate or product). Among them PaDADH Glu246Gln, showing an increased product release rate,turned out to be a faster and better performing biocatalyst for industrial applications. Overall, this study demonstrates that the proximity of a gating residue to the active site and interactions with ligands are two important factors to consider during protein engineering.

Expanded Substrate Specificity in D-Amino Acid Transaminases: A Case Study of Transaminase from Blastococcus saxobsidens.
Shilova SA, Matyuta IO, Petrova ES, Nikolaeva AY, Rakitina TV, Minyaev ME, Boyko KM, Popov VO, Bezsudnova EY. Int J Mol Sci. 2023 Nov 10;24(22):16194. doi: 10.3390/ijms242216194.

In this paper, a detailed analysis of the structure/function relationships of a non-canonical, PLP-dependent, D-amino acid transaminase active on D-amino acids, α-keto acids, and primary aromatic (R)-amines is reported. Authors suggest that structural determinants in the active site facilitate the binding of substrates with and without α-carboxylate groups. This expanded substrate specificity makes this enzyme a good starting point for the design of biocatalysts with new substrate specificity for the industrial stereoselective amination of various compounds.

A newly identified enzyme from Japanese common squid Todarodes pacificus has the ability to biosynthesize D-aspartate.
Koyama H, Takahashi Y, Matori S, Kuniyoshi H, Kurose K. Arch Biochem Biophys. 2023 Dec;750:109809. doi: 10.1016/

In this study, Koyama and his collaborators successfully identified and charcaterized a novel gene encoding aspartate racemase (AspRase). Intriguingly, AspRase turned to be a unique enzyme being structurally and phylogenetically related to aspartate aminotransferase (AST), rather than similar to other known AspRases, and possessing both AspRase and AST activities. The enzyme can biosynthesize D-Asp and L-Glu from L-Asp and 2-oxoglutarate, with L- Glu being synthesized slightly more than D-Asp. As highlighted by the authors, this study provides insights into the D-Asp metabolic pathway in the cephalopod nervous system, but also suggests an alternative strategy for the identification of AspRase in mammals: the enzyme responsible for D-aspartate biosynthesis therein has not been clarified yet, but it is likely that another AST-related enzyme is involved.

On the regulation of human D-aspartate oxidase.
Rabattoni V, Motta Z, Miceli M, Molla G, Fissore A, Adinolfi S, Pollegioni L, Sacchi S. Protein Sci. 2023 Nov;32(11):e4802. doi: 10.1002/pro.4802.

This piece of work provides for the first time an insight into the regulation of human D-aspartate oxidase (hDASPO), and therefore D-aspartate catabolism. The authors focused on post-translational modification and protein-protein interaction and, combining in vitro and cellular studies, revealed that the enzyme can be nitrosylated in vitro, although the modification affects its activity to a limited extent. Most notably, hDASPO was shown to interact with pLG72, a small primate specific protein known to modulate the functional properties of the homologous enzyme D-amino acid oxidase (hDAAO). In line with the proposed role as a negative chaperone affecting the enzyme activity and stability, pLG72 promotes hDASPO inactivation in vitro, while at the cellular level its coexpression negatively affects hDASPO level by reducing its half-life. Based on these findings, hDASPO:pLG72 complex formation was suggested to represent a safety mechanism to avoid cytotoxicity due to H2O2
produced by hDASPO catalysed reaction.

Characterization of E121K mutation of D-amino acid oxidase – Insights into mechanisms
leading to amyotrophic lateral sclerosis.
Dave U, Khan S, Gomes J. (2023) Biochim Biophys Acta Proteins Proteom. 1871(6):140947.
doi: 10.1016/j.bbapap.2023.140947.

Here the characterization of a variant of D-amino acid oxidase (D-serine degrading enzyme) carrying a new, low incidence mutation, Glu121Lys, identified in a cohort of ALS patients from India is reported. As demonstrated for other ALS-related mutations in this flavoenzyme, E121K substitution was found to largely inactivate the enzyme activity and profoundly affect both the cofactor and substrate binding properties, and the protein secondary and tertiary structure. In particular, E121K- DAO variant showed higher beta-sheet content compared to the wild-type and was remarkably prone to aggregation that proceeds through the intermediate formation of fibrillar structures. Altogether, these findings suggested E121K-DAO potential role in ALS pathogenesis: the enzyme loss of function promote toxicity and cell death due either to the accumulation of D-Ser and the formation of aggregates.





Occurrence of D-amino acids in natural products.
Armstrong DW, Berthod A. Nat Prod Bioprospect. 2023 Nov 7;13(1):47. doi: 10.1007/s13659-

 D-amino acids are found in numerous natural peptide compounds produced mainly by bacteria, algae, fungi, or marine animals, but also by vertebrates. This comprehensive review focuses on what is their origin, how were they identified, and what is their function.





Two-dimensional LC-MS/MS and three-dimensional LC analysis of chiral amino acids and related compounds in real-world matrices.
Ishii C, Hamase K. J Pharm Biomed Anal. 2023 Oct 25;235:115627. doi:
10.1016/j.jpba.2023.115627. Epub 2023 Aug 8. PMID: 37633168.

 This is a truly complete review, written by experts in field, that deal with multi-dimensional LC and LC/MS techniques, namely, 3D-HPLC(LC/LC/LC) and 2D LC-MS/MS (LC/LC/MS/MS), and their possible applications. They are very powerful analytical methods allowing the detection of trace amounts of amino acid enantiomers (in the free form or present in peptides and proteins) in a wide variety of biological samples, ranging from body fluids for clinical analysis to beverages and foodstuff, and even in extra-terrestrial samples.

Analysis of D-amino acids secreted from murine islets of Langerhans using Marfey’s reagent and reversed phase LC-MS/MS.
Ogunkunle EO, Davis JJ, Skinner EL, Thornham J, Roper MG. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Nov 10;1231:123928. doi: 10.1016/j.jchromb.2023.123928.

Here, a comprehensive assessment of D-amino acids (D-AAs) in murine islets of Langerhans secretions is provided. Authors use a selective LC-MS/MS method based on 1-fluoro-2,4- dinitrophenyl-5-L-alanine amide (Marfey’s reagent, MR) as a derivatizing reagent for chiral amino acid separations, to resolve the various D-AAs present under different stimulation conditions. D-serine and D-glutamine, but not D-aspartate were detected in secretions from islets of Langerhans after glucose stimulation. Taken together, these findings, coupled with the previously reported presence of serine racemase and N-methyl-D-aspartate receptors, suggest a potential role for D-AAs as signaling molecules in this endocrine tissue.

Separation and Identification of Isoleucine Enantiomers and Diastereomers Using an Original Chiral Resolution Labeling Reagent
Makoto Ozaki, Motoshi Shimotsuma, Takefumi Kuranaga, Hideaki Kakeya, Tsunehisa Hirose
Chem Pharm Bull (Tokyo). 2023 Aug 24. doi: 10.1248/cpb.c23-00439. Online ahead of print.

Among the 20 amino acids that make up proteins, threonine (Thr) and isoleucine (Ile) have two chiral carbons and thus have two enantiomers and diastereomers. The separation and identification of these stereoisomers by HPLC are laborious and complicated. Here, an analytical method for the separation and identification of Ile stereoisomers through liquid chromatography-mass spectrometry based on a chiral resolution labeling reagent (1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine-amide, L-FDVDA) and a PBr column packed with pentabromobenzyl-modified silica gel was set up. Twenty D,L-amino acids including Thr stereoisomers (41 amino acids including glycine) were separated and identified using C18 column, while Ile stereoisomers were separated using a PBr column. Additionally, peptides containing Thr and Ile stereoisomers can be accurately detected through labeling with L-FDVDA.

Variable fragmentation and ionization of amyloid-beta epimers and isomers.
Readel ER, Dhaubhadel U, Patel A, Armstrong DW. Anal Bioanal Chem. 2023
Nov;415(27):6799-6807. doi: 10.1007/s00216-023-04958-3.

Despite, a elevated presence of epimeric and isomeric amino acids modifications (i.e. D-antipode and/or L-D-iso-Asp) at all Asp and Ser positions were observed in the Amyloid-beta (Aβ) protein extracted from AD patients, quantitation and identification of the exact location of these “aberrant” amino acids have been problematic. Recently, methods for selectively identifying modified peptides extracted from AD patients were developed by combing chiral stationary phases with HPLC. The sensitivity of these methods was enhanced by using mass spectrometry detection. The goal of this work was to evaluate the epimeric and/or isomeric tryptic fragments of Aβ peptide to determine if they are uniquely affected and detected by MS/MS.





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).

The guiding principle is support the research projects aimed to investigate the involvement of D-amino acids in main physiological processes, from bacteria to humans. The ultimate goal is to actively participate to the elucidation of the mechanisms by which the D-amino acids perform specific functions, and to identify their presence and concentration in different organisms and compartments, also with regards to well-established functional states, with particular emphasis to pathological states. Understand the involvement of D-amino acids in important diseases as a way to set up novel therapeutic strategies.




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