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The human flavoenzyme D-aspartate oxidase (hDASPO) controls the level of D-aspartate in the brain, a molecule acting as an agonist of NMDA receptors and modulator of AMPA and mGlu5 receptors. hDASPO-induced D-aspartate degradation prevents age-dependent deterioration of brain functions and is related to psychiatric disorders such as schizophrenia and autism. Notwithstanding this crucial role, less is known about hDASPO regulation. Here, we report that hDASPO is nitrosylated in vitro, while no evidence of sulfhydration and phosphorylation is apparent: nitrosylation affects the activity of the human flavoenzyme to a limited extent. Furthermore, hDASPO interacts with the primate-specific protein pLG72 (a well-known negative chaperone of D-amino acid oxidase, the enzyme deputed to D-serine degradation in the human brain), yielding a ~114 kDa complex, with a micromolar dissociation constant, promoting the flavoenzyme inactivation. At the cellular level, pLG72 and hDASPO generate a cytosolic complex: the expression of pLG72 negatively affects the hDASPO level by reducing its half-life. We propose that pLG72 binding may represent a protective mechanism aimed at avoiding cytotoxicity due to H 2 O 2 produced by the hDASPO enzymatic degradation of D-aspartate, especially before the final targeting to peroxisomes., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. l-Serine is the precursor of the two main co-agonists of the N-methyl-d-aspartate receptor, glycine and d-serine. Strikingly, dysfunctions in both l- and d-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l-serine, glycine, threonine, l- and d-aspartate (which level is unexpectedly higher than that of d-serine) show the same biosynthetic time course. A significant utilization of l-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d-serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In humans, the phosphorylated pathway (PP) converts the glycolytic intermediate D-3-phosphoglycerate (3-PG) into L-serine through the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase. From the pathogenic point of view, the PP in the brain is particularly relevant, as genetic defects of any of the three enzymes are associated with a group of neurometabolic disorders known as serine deficiency disorders (SDDs). We recombinantly expressed and characterized eight variants of PSAT associated with SDDs and two non-SDD associated variants. We show that the pathogenetic mechanisms in SDDs are extremely diverse, including low affinity of the cofactor pyridoxal 5'-phosphate and thermal instability for S179L and G79W PSAT, loss of activity of the holo form for R342W PSAT, aggregation for D100A PSAT, increased K m for one of the substrates with invariant k cat s for S43R PSAT, and a combination of increased K m and decreased k cat for C245R PSAT. Finally, we show that the flux through the in vitro reconstructed PP at physiological concentrations of substrates and enzymes is extremely sensitive to alterations of the functional properties of PSAT variants, confirming PSAT dysfunctions as a cause of SSDs.

De novo l-serine biosynthesis in the mammalian astrocytes proceeds via a linear, three-step pathway (the phosphorylated pathway) catalysed by 3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP). The first reaction, catalysed by PHGDH and using the glycolytic intermediate 3-phosphoglycerate, is strongly shifted towards the reagents, and coupling to the following step by PSAT is required to push the equilibrium towards l-serine formation; the last step, catalysed by PSP, is virtually irreversible and inhibited by the final product l-serine. Very little is known about the regulation of the human phosphorylated pathway and the ability of the three enzymes to organise in a complex with potential regulatory functions. Here, the complex formation was investigated in differentiated human astrocytes, by proximity ligation assay, and in vitro on the human recombinant enzymes. The results indicate that the three enzymes co-localise in cytoplasmic clusters that more stably engage PSAT and PSP. Although in vitro analyses based on native PAGE, size exclusion chromatography and cross-linking experiments do not show the formation of a stable complex, kinetic studies of the reconstituted pathway using physiological enzyme and substrate concentrations support cluster formation and indicate that PHGDH catalyses the rate-limiting step while PSP reaction is the driving force for the whole pathway. The enzyme agglomerate assembly of the phosphorylated pathway (the putative 'serinosome') delivers a relevant level of sophistication to the control of l-serine biosynthesis in human cells, a process strictly related to the modulation of the brain levels of d-serine and glycine, the main co-agonists of N-methyl-d-aspartate receptors and various pathological states., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The 'enzyme prodrug therapy' represents a promising strategy to overcome limitations of current cancer treatments by the systemic administration of prodrugs, converted by a foreign enzyme into an active anticancer compound directly in tumor sites. One example is D-amino acid oxidase (DAAO), a dimeric flavoenzyme able to catalyze the oxidative deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS), able to favor cancer cells death. A DAAO variant containing five aminoacidic substitutions (mDAAO) was demonstrated to possess a better therapeutic efficacy under low O 2 concentration than wild-type DAAO (wtDAAO). Recently, aiming to design promising nanocarriers for DAAO, multi-walled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG) to reduce their tendency to aggregation and to improve their biocompatibility. Here, wtDAAO and mDAAO were adsorbed on PEGylated MWCNTs and their activity and cytotoxicity were tested. While PEG-MWCNTs-DAAOs have shown a higher activity than pristine MWCNTs-DAAO (independently on the DAAO variant used), PEG-MWCNTs-mDAAO showed a higher cytotoxicity than PEG-MWCNTs-wtDAAO at low O 2 concentration. In order to evaluate the nanocarriers' biocompatibility, PEG-MWCNTs-DAAOs were incubated in human serum and the composition of protein corona was investigated via nLC-MS/MS, aiming to characterize both soft and hard coronas. The mDAAO variant has influenced the bio-corona composition in both number of proteins and presence of opsonins and dysopsonins: notably, the soft corona of PEG-MWCNTs-mDAAO contained less proteins and was more enriched in proteins able to inhibit the immune response than PEG-MWCNTs-wtDAAO. Considering the obtained results, the PEGylated MWCNTs conjugated with the mDAAO variant seems a promising candidate for a selective antitumor oxidative therapy: under anoxic-like conditions, this novel drug delivery system showed a remarkable cytotoxic effect controlled by the substrate addition, against different tumor cell lines, and a bio-corona composition devoted to prolong its blood circulation time, thus improving the drug's biodistribution., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03568-1., Competing Interests: Conflict of interestThe authors declare that there is no competing conflict of interest., (© The Author(s) 2023.)

Enzymatic degradation of poly(ethylene terephthalate) (PET) is becoming a reality because of the identification of novel PET-hydrolysing enzymes (PHEs) and the engineering of evolved enzyme variants. Here, improved variants of leaf-branch compost cutinase (LCC), a thermostable enzyme isolated by a metagenomic approach, were generated by a semi-rational protein engineering approach. Starting from a deleted LCC form lacking the secretion signal (ΔLCC), single and double variants possessing a higher activity on PET were isolated. The single-point F243T ΔLCC variant partially (~ 67%) depolymerized amorphous PET film producing ~ 21.9 mm of products after 27 h of reaction at 72 °C. The S101N/F243T ΔLCC double variant reached a further increase in activity on PET. Notably, for both single and double variants the highest conversion yield was obtained at 55 °C. Kinetics studies and molecular dynamics simulations support that a slight decreased affinity for PET is responsible for the superior degradation performance of the S101N/F243T variant and that this stems from a slightly higher flexibility of the active site region close to position 243. Furthermore, our findings question the need for a high reaction temperature for PET degradation, at least for LCC: at ≥ 70 °C, the conversion of amorphous PET into a more crystalline polymer, resistant to enzymatic hydrolysis, is favoured. The evolved S101N/F243T ΔLCC variant is able to depolymerize fully 1.3 g of untreated postconsumer PET waste in ≤ 3 days at 55 °C (using 1.25 mg of enzyme only), this making PET enzymatic degradation by engineering LCC a more ecofriendly and sustainable process., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (≥99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines.

Chinese Hamster Ovary cells (CHO) have become the most common workhorse for the commercial production of therapeutic proteins, as well as for the production of recombinant proteins for biomedical research. The ability to grow at high density in suspension, the adaptability to serum free media, and the ease transfection and scale up, made CHO cell line highly productive and robust for large-scale production. Here, we present an optimized workflow used to successfully express and purify a number of human proteins with a yield up to 5 mg/L of culture. The entire protocol, from the synthetic gene design to the assessment of purified protein quality, can be completed in 2 weeks. The established cell culture platform has been efficiently adapted to rapidly produce the receptor-binding domain (RBD) in SARS-CoV-2 S protein, a protein required by many laboratories in 2020 to better understand the initial step of infection related to COVID-19 pandemic. An overall yield of 2 mg of high quality soluble RBD per liter of culture was obtained, a production 10-times cheaper than commercial preparations, this representing an intriguing strategy for future challenges., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

Organisms from all kingdoms of life synthesize L-serine (L-Ser) from 3-phosphoglycerate through the phosphorylated pathway, a three-step diversion of glycolysis. Phosphoserine aminotransferase (PSAT) catalyzes the intermediate step, the pyridoxal 5'-phosphate-dependent transamination of 3-phosphohydroxypyruvate and L-glutamate to O-phosphoserine (OPS) and α-ketoglutarate. PSAT is particularly relevant in the central nervous system of mammals because L-Ser is the metabolic precursor of D-serine, cysteine, phospholipids, and nucleotides. Several mutations in the human psat gene have been linked to serine deficiency disorders, characterized by severe neurological symptoms. Furthermore, PSAT is overexpressed in many tumors and this overexpression has been associated with poor clinical outcomes. Here, we report the detailed functional and structural characterization of the recombinant human PSAT. The reaction catalyzed by PSAT is reversible, with an equilibrium constant of about 10, and the enzyme is very efficient, with a k cat /K m of 5.9 × 10 6  M -1  s -1 , thus contributing in driving the pathway towards the products despite the extremely unfavorable first step catalyzed by 3-phosphoglycerate dehydrogenase. The 3D X-ray crystal structure of PSAT was solved in the substrate-free as well as in the OPS-bound forms. Both structures contain eight protein molecules in the asymmetric unit, arranged in four dimers, with a bound cofactor in each subunit. In the substrate-free form, the active site of PSAT contains a sulfate ion that, in the substrate-bound form, is replaced by the phosphate group of OPS. Interestingly, fast crystal soaking used to produce the substrate-bound form allowed the trapping of different intermediates along the catalytic cycle., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Microbial communities inhabiting the Antarctic Ocean show psychrophilic and halophilic adaptations conferring interesting properties to the enzymes they produce, which could be exploited in biotechnology and bioremediation processes. Use of cold- and salt-tolerant enzymes allows to limit costs, reduce contaminations, and minimize pretreatment steps. Here, we report on the screening of 186 morphologically diverse microorganisms isolated from marine biofilms and water samples collected in Terra Nova Bay (Ross Sea, Antarctica) for the identification of new laccase activities. After primary screening, 13.4 and 10.8% of the isolates were identified for the ability to oxidize 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and the dye azure B, respectively. Amongst them, the marine Halomonas sp. strain M68 showed the highest activity. Production of its laccase-like activity increased six-fold when copper was added to culture medium. Enzymatic activity-guided separation coupled with mass spectrometry identified this intracellular laccase-like protein (named Ant laccase) as belonging to the copper resistance system multicopper oxidase family. Ant laccase oxidized ABTS and 2,6-dimethoxy phenol, working better at acidic pHs The enzyme showed a good thermostability, with optimal temperature in the 40-50°C range and maintaining more than 40% of its maximal activity even at 10°C. Furthermore, Ant laccase was salt- and organic solvent-tolerant, paving the way for its use in harsh conditions. To our knowledge, this is the first report concerning the characterization of a thermo- and halo-tolerant laccase isolated from a marine Antarctic bacterium., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bisaccia, Binda, Rosini, Caruso, Dell'Acqua, Azzaro, Laganà, Tedeschi, Maffioli, Pollegioni and Marinelli.)

α-amino acids exist in two configurations, named D-( dextro ) and L-( levo ) enantiomers. L-amino acids are used in protein synthesis and play a central role in cell metabolism. The effects of the L-amino acid composition of foods and the dietary modifications of this composition on the efficacy of cancer therapies have been widely investigated in relation to the growth and reproduction of cancerous cells. However, less is known about the involvement of D-amino acids. In recent decades, D-amino acids have been identified as natural biomolecules that play interesting and specific roles as common components of the human diet. Here, we focus on recent investigations showing altered D-amino acid levels in specific cancer types and on the various roles proposed for these biomolecules related to cancer cell proliferation, cell protection during therapy, and as putative, innovative biomarkers. Notwithstanding recent progress, the relationship between the presence of D-amino acids, their nutritional value, and cancer cell proliferation and survival represents an underrated scientific issue. Few studies on human samples have been reported to date, suggesting a need for routine analysis of D-amino acid content and an evaluation of the enzymes involved in regulating their levels in clinical samples in the near future.

Invited for this month's cover is the work by Claudia Crestini and collaborators at Ca'Foscari University of Venice, Italy, and University of Insubria, Italy. The image shows the formation of low-molecular-weight compounds by the oxidative depolymerization of lignin by the laccase-Lig multienzymatic multistep system. The Research Article itself is available at 10.1002/cssc.202201147., (© 2022 Wiley-VCH GmbH.)

The oxidation therapy, based on the controlled production of Reactive Oxygen Species directly into the tumor site, was introduced as alternative antitumor approach. For this purpose, d-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, an enzyme able to efficiently catalyze the production of hydrogen peroxide from d-amino acids, was adsorbed onto multi-walled carbon nanotubes (MWCNTs), previously functionalized with polylactic-co-glycolic acid (PLGA) or polyethylene glycol (PEG) at different degrees to reduce their toxicity, to be targeted directly into the tumor. In vitro activity and cytotoxicity assays demonstrated that DAAO-functionalized nanotubes (f-MWCNTs) produced H2O2 and induced toxic effects to selected tumor cell lines. After incubation in human plasma, the protein corona was investigated by SDS-PAGE and mass spectrometry analysis. The enzyme nanocarriers generally seemed to favor their biocompatibility, promoting the interaction with dysopsonins. Despite this, PLGA or high degree of PEGylation promoted the adsorption of immunoglobulins with a possible activation of immune response and this effect was probably due to PLGA hydrophobicity and dimensions and to the production of specific antibodies against PEG. In conclusion, the PEGylated MWCNTs at low degree seemed the most biocompatible nanocarrier for adsorbed DAAO, preserving its anticancer activity and forming a bio-corona able to reduce both defensive responses and blood clearance., (© 2022. The Author(s).)

d-Amino acids (d-AAs) are key components of the peptidoglycan matrix in bacterial cells. Various bacterial species are known to produce d-AAs by using different enzymes, such as highly specific and broad-spectrum racemases. Miyamoto et al. studied the biosynthesis of d-glutamate in the hyperthermophile and anaerobic Gram-negative bacterium, Thermotoga maritima, which does not possess a broad-spectrum racemase. The investigated TM0831 enzyme catalyzes both a d-amino acid aminotransferase reaction producing d-glutamate and an amino acid racemase activity aimed at generating d-aspartate and d-glutamate from the corresponding l-enantiomers. TM0831 represents an example of natural molecular evolution process favoring the enzyme versatility. Comment on: https://doi.org/10.1111/febs.16452., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Healthy aging is an ambitious aspiration for humans, but neurodegenerative disorders, such as Alzheimer's disease (AD), strongly affect quality of life. Using an integrated omics approach, we investigate alterations in the molecular composition of postmortem hippocampus samples of healthy persons and individuals with AD. Profound differences are apparent between control and AD male and female cohorts in terms of up- and downregulated metabolic pathways. A decrease in the insulin response is evident in AD when comparing the female with the male group. The serine metabolism (linked to the glycolytic pathway and generating the N-methyl-D-aspartate [NMDA] receptor coagonist D-serine) is also significantly modulated: the D-Ser/total serine ratio represents a way to counteract age-related cognitive decline in healthy men and during AD onset in women. These results show how AD changes and, in certain respects, almost reverses sex-specific proteomic and metabolomic profiles, highlighting how different pathophysiological mechanisms are active in men and women., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In 2002, the novel human gene G72 was associated with schizophrenia susceptibility. This gene encodes a small protein of 153 amino acids, named pLG72, which represents a rare case of primate-specific protein. In particular, the rs2391191 single nucleotide polymorphism (resulting in in the R30K substitution) was robustly associated to schizophrenia and bipolar disorder. In this review, we aim to summarize the results of 20 years of biochemical investigations on pLG72. The main known role of pLG72 is related to its ability to bind and inactivate the flavoenzyme d-amino acid oxidase, i.e., the enzyme that controls the catabolism of d-serine, the main NMDA receptor coagonist in the brain. pLG72 was proposed to target the cytosolic form of d-amino acid oxidase for degradation, preserving d-serine and protecting the cell from oxidative stress generated by hydrogen peroxide produced by the flavoenzyme reaction. Anyway, pLG72 seems to play additional roles, such as affecting mitochondrial functions. The level of pLG72 in the human body is still a controversial issue because of its low expression and challenging detection. Anyway, the intriguing hypothesis that pLG72 level in blood could represent a suitable marker of Alzheimer's disease progression (a suggestion not sufficiently established yet) merits further investigations.

A number of approaches have been developed over the years to manage cancer, such as chemotherapy using low-molecular-mass molecules and radiotherapy. Here, enzymes can also find useful applications. Among them, oxidases have attracted attention because of their ability to produce reactive oxygen species (ROS, especially hydrogen peroxide) in tumors and potentially modulate the production of this cytotoxic compound when enzymes active on substrates present in low amounts are used, such as the d-amino acid oxidase and d-amino acid couple system. These treatments have been also developed for additional cancer treatment approaches, such as phototherapy, nutrient starvation, and metal-induced hydroxyl radical production. In addition, to improve tumor specificity and decrease undesired side effects, oxidases have been targeted by means of nanotechnologies and protein engineering (i.e., by designing chimeric proteins able to accumulate in the tumor). The most recent advances obtained by using six different oxidases (i.e., the FAD-containing enzymes glucose oxidase, d- and l-amino acid oxidases, cholesterol oxidase and xanthine oxidase, and the copper-containing amine oxidase) have been reported. Anticancer therapy based on oxidase-based ROS production has now reached maturity and can be applied in the clinic., (© 2021 International Union of Biochemistry and Molecular Biology.)

Background: D-amino acids are present in the human body originating from diet, bacterial flora, and endogenous synthesis (at least for D-serine and, probably, D-aspartate). D-amino acids are involved in important physiological processes (e.g., D-serine and D-aspartate act on the N-methyl-D-aspartate receptor as co-agonist and agonist, respectively) and increasing evidence links D-amino acids to different pathological states., Methods: Determination of D-amino acids levels in blood is mainly based on enantiomeric separations by high performance liquid chromatography. Because of the low amount of D-enantiomers compared to the corresponding L-amino acids and the high background noise associated with biological matrices, positive and negative controls are absolutely required to obtain reliable values., Results: Altered levels of D-serine in blood have been reported in several neurological and psychiatric disorders: it has been proposed as promising biomarker in schizophrenia, Alzheimer's disease, and amyotrophic lateral sclerosis. Indeed, D-serine levels seem an appropriate predictor of anti-depressant response in major depressive disorder and posttraumatic stress disorder, as well as a prognostic biomarker of early cognitive decline, especially when considering D-serine and D-proline levels simultaneously. Furthermore, D-amino acids seem useful biomarkers for pathologies not related to the central nervous system, such as pancreatic cancer and chronic kidney diseases., Conclusion: This is the first review focusing on the determination of blood levels of Damino acids as diagnostic and prognostic biomarkers. The experimental evidence of involvement of D-amino acids in various physiological pathways suggest investigating their levels in additional pathologies too, such as diabetes mellitus. In conclusion, the levels of D-amino acids in blood may represent novel diagnostic peripheral biomarkers for various disorders. Further studies are required to standardize/automatize the determinations and for confirming their clinical effectiveness., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-ΔIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-ΔIsPET variant. By using 0.1 mg mL -1 of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL -1 PET microparticles (~26% depolymerization yield). Indeed, TS-ΔIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 μg of enzyme mL -1 . The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation.

Glycine is an important biomarker in clinical analysis due to its involvement in multiple physiological processes. As such, the need for low-cost analytical tools for glycine detection is growing. As a neurotransmitter, glycine is involved in inhibitory and excitatory neurochemical transmission in the central nervous system. In this work, we present a 10 μM Pt-based electrochemical enzymatic biosensor based on the flavoenzyme glycine oxidase (GO) for localized real-time measurements of glycine. Among GO variants at position 244, the H244K variant with increased glycine turnover was selected to develop a functional biosensor. This biosensor relies on amperometric readouts and does not require additional redox mediators. The biosensor was characterized and applied for glycine detection from cells, mainly HEK 293 cells and primary rat astrocytes. We have identified an enzyme, GO H244K, with increased glycine turnover using mutagenesis but which can be developed into a functional biosensor. Noteworthy, a glycine release of 395.7 ± 123 μM from primary astrocytes was measured, which is ∼fivefold higher than glycine release from HEK 293 cells (75.4 ± 3.91 μM) using the GO H244K biosensor.

Challenges facing enzyme-based electrochemical sensors include substrate specificity, batch to batch reproducibility, and lack of quantitative metrics related to the effect of enzyme immobilization. We present a quick, simple, and general approach for measuring the effect of immobilization and cross-linking on enzyme activity and substrate specificity. The method can be generalized for electrochemical biosensors using an enzyme that releases hydrogen peroxide during its catalytic cycle. Using as proof of concept RgDAAO-based electrochemical biosensors, we found that the Michaelis-Menten constant (K m ) decreases post immobilization, hinting at alterations in the enzyme kinetic properties and thus substrate specificity. We confirm the decrease in K m electrochemically by characterizing the substrate specificity of the immobilized RgDAAO using chronoamperometry. Our results demonstrate that enzyme immobilization affects enzyme substrate specificity and this must be carefully evaluated during biosensor development., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Human d-aspartate oxidase (hDASPO) is a FAD-dependent enzyme responsible for the degradation of d-aspartate (d-Asp). In the mammalian central nervous system, d-Asp behaves as a classical neurotransmitter, it is thought to be involved in neural development, brain morphology and behavior, and appears to be involved in several pathological states, such as schizophrenia and Alzheimer's disease. Apparently, the human DDO gene produces alternative transcripts encoding for three putative hDASPO isoforms, constituted by 341 (the 'canonical' form), 369, and 282 amino acids. Despite the increasing interest in hDASPO and its physiological role, little is known about these different isoforms. Here, the additional N-terminal peptide present in the hDASPO_369 isoform only has been identified in hippocampus of Alzheimer's disease female patients, while peptides corresponding to the remaining part of the protein were present in samples from male and female healthy controls and Alzheimer's disease patients. The hDASPO_369 isoform was largely expressed in E. coli as insoluble protein, hampering with its biochemical characterization. Furthermore, we generated U87 human glioblastoma cell clones stably expressing hDASPO_341 and, for the first time, hDASPO_369 isoforms; the latter protein showed a lower expression compared with the canonical isoform. Both protein isoforms are active (showing similar kinetic properties), localize to the peroxisomes, are very stable (a half-life of approximately 100 h has been estimated), and are primarily degraded through the ubiquitin-proteasome system. These studies shed light on the properties of hDASPO isoforms with the final aim to clarify the mechanisms controlling brain levels of the neuromodulator d-Asp., (© 2021 Federation of European Biochemical Societies.)

The polyester PET (poly(ethylene terephthalate)) plastic is chemically inert and remarkably persistent, posing relevant and global pollution concerns due to its accumulation in ecosystems across the globe. In past years, research focused on identifying bacteria active on PET and on the specific enzymes responsible for its degradation. Here, the enzymatic degradation of PET can be considered as an 'erosion process' that takes place on the surface of an insoluble material and results in an unusual, substrate-limited kinetic condition. In this review, we report on the most suitable models to evaluate the kinetics of PET-hydrolyzing enzymes, which takes into consideration the amount of enzyme adsorbed on the substrate, the enzyme-accessible ester bonds, and the product inhibition effects. Careful kinetic analysis is especially relevant to compare enzymes from different sources and evolved variants generated by protein engineering studies as well. Furthermore, the analytical methods most suitable to screen natural bacteria and recombinant variant libraries generated by protein engineering have been also reported. These methods rely on different detection systems and are performed both on model compounds and on different PET samples (e.g., nanoparticles, microparticles, and waste products). All this meaningful information represents an optimal starting point and boosts the process of identifying systems able to biologically recycle PET waste products., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

d-Amino acid oxidase (DAAO) enzymes bind a range of d-amino acids with variable affinity. As such, the design of selective DAAO-based enzymatic biosensors remains a challenge for real-world biosensor application. Herein, a methodology for developing biosensors with varying substrate selectivity is presented. First, we address DAAO-based biosensor selectivity toward d-serine by introducing point mutations into DAAO using rational design. Next, the wild-type yeast DAAO ( Rg DAAO WT) and variants human DAAO W209R and yeast M213G are characterized for their selectivity and activity toward d-serine and d-alanine, the preferred DAAO substrates. The DAAO enzymes have been immobilized for final biosensor design, where they demonstrate selectivity comparable to free DAAO. The cross-linking procedure impacts on DAAO structure and function and the use of a regeneration strategy allows the biosensor response to be improved.

In recent years, the D-enantiomers of amino acids have been recognized as natural molecules present in all kingdoms, playing a variety of biological roles. In humans, d-serine and d-aspartate attracted attention for their presence in the central nervous system. Here, we focus on d-aspartate, which is involved in glutamatergic neurotransmission and the synthesis of various hormones. The biosynthesis of d-aspartate is still obscure, while its degradation is due to the peroxisomal flavin adenine dinucleotide (FAD)-containing enzyme d-aspartate oxidase. d-Aspartate emergence is strictly controlled: levels decrease in brain within the first days of life while increasing in endocrine glands postnatally and through adulthood. The human d-aspartate oxidase (hDASPO) belongs to the d-amino acid oxidase-like family: its tertiary structure closely resembles that of human d-amino acid oxidase (hDAAO), the enzyme that degrades neutral and basic d-amino acids. The structure-function relationships of the physiological isoform of hDASPO (named hDASPO_341) and the regulation of gene expression and distribution and properties of the longer isoform hDASPO_369 have all been recently elucidated. Beyond the substrate preference, hDASPO and hDAAO also differ in kinetic efficiency, FAD-binding affinity, pH profile, and oligomeric state. Such differences suggest that evolution diverged to create two different ways to modulate d-aspartate and d-serine levels in the human brain. Current knowledge about hDASPO is shedding light on the molecular mechanisms underlying the modulation of d-aspartate levels in human tissues and is pushing novel, targeted therapeutic strategies. Now, it has been proposed that dysfunction in NMDA receptor-mediated neurotransmission is caused by disrupted d-aspartate metabolism in the nervous system during the onset of various disorders (such as schizophrenia): the design of suitable hDASPO inhibitors aimed at increasing d-aspartate levels thus represents a novel and useful form of therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pollegioni, Molla, Sacchi and Murtas.)

Prefrontal control of cognitive functions critically depends upon glutamatergic transmission and N-methyl D-aspartate (NMDA) receptors, the activity of which is regulated by dopamine. Yet whether the NMDA receptor coagonist d-serine is implicated in the dopamine-glutamate dialogue in the prefrontal cortex (PFC) and other brain areas remains unexplored. Here, using electrophysiological recordings, we show that d-serine is required for the fine-tuning of glutamatergic neurotransmission, neuronal excitability, and synaptic plasticity in the PFC through the actions of dopamine at D 1 and D 3 receptors. Using in vivo microdialysis, we show that D 1 and D 3 receptors exert a respective facilitatory and inhibitory influence on extracellular levels and activity of d-serine in the PFC, with actions expressed primarily via the cAMP/protein kinase A (PKA) signaling cascade. Further, using functional magnetic resonance imaging (fMRI) and behavioral assessment, we show that d-serine is required for the potentiation of cognition by D 3 R blockade as revealed in a test of novel object recognition memory. Collectively, these results unveil a key role for d-serine in the dopaminergic neuromodulation of glutamatergic transmission and PFC activity, findings with clear relevance to the pathogenesis and treatment of diverse brain disorders involving alterations in dopamine-glutamate cross-talk., Competing Interests: Competing interest statement: M.J.M. and J.-M.R., respectively, were and are full-time employees of Institut de Recherche Servier and have no other interests to declare. S. Schmidt and J.K. are employees of Pronexus Analytical AB but declare no conflicts of interests.

In the central nervous system, the flavoprotein D-amino acid oxidase is responsible for catabolizing D-serine, the main endogenous coagonist of N-methyl-D-aspartate receptor. Dysregulation of D-serine brain levels in humans has been associated with neurodegenerative and psychiatric disorders. This D-amino acid is synthesized by the enzyme serine racemase, starting from the corresponding L-enantiomer, and degraded by both serine racemase (via an elimination reaction) and the flavoenzyme D-amino acid oxidase. To shed light on the role of human D-amino acid oxidase (hDAAO) in D-serine metabolism, the structural/functional relationships of this enzyme have been investigated in depth and several strategies aimed at controlling the enzymatic activity have been identified. Here, we focused on the effect of post-translational modifications: by using a combination of structural analyses, biochemical methods, and cellular studies, we investigated whether hDAAO is subjected to nitrosylation, sulfhydration, and phosphorylation. hDAAO is S-nitrosylated and this negatively affects its activity. In contrast, the hydrogen sulfide donor NaHS seems to alter the enzyme conformation, stabilizing a species with higher affinity for the flavin adenine dinucleotide cofactor and thus positively affecting enzymatic activity. Moreover, hDAAO is phosphorylated in cerebellum; however, the protein kinase involved is still unknown. Taken together, these findings indicate that D-serine levels can be also modulated by post-translational modifications of hDAAO as also known for the D-serine synthetic enzyme serine racemase., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sacchi, Rabattoni, Miceli and Pollegioni.)

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD + /NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.

The flavoenzyme D-amino acid oxidase (DAAO) is deputed to the degradation of D-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal D-amino acids to generate hydrogen peroxide (H 2 O 2 ), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1-16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

D-Serine acts as a co-agonist of N-methyl-D-aspartate receptors (NMDAR) which appear overactivated in AD, while D-aspartate is a modulatory molecule acting on NMDAR as a second agonist. The aim of this work is to clarify whether the levels of these D-amino acids in serum are deregulated in AD, with the final goal to identify novel and precocious biomarkers in AD. Serum levels of L- and D-enantiomers of serine and aspartate were determined by HPLC using a pre-column derivatization procedure and a selective enzymatic degradation. Experimental data obtained from age-matched healthy subjects (HS) and AD patients were statistically evaluated by considering age, gender, and disease progression, and compared. Minor changes were apparent in the serum L- and D-aspartate levels in AD patients compared to HS. A positive correlation for the D-serine level and age was apparent in the AD cohort. Notably, the serum D-serine level and the D-/total serine ratio significantly increased with the progression of the disease. Gender seems to have a minor effect on the levels of all analytes tested. This work proposes that the serum D-serine level and D-/total serine ratio values as novel and valuable biomarkers for the progression of AD: the latter parameter allows to discriminate CDR 2 and CDR 1 patients from healthy (CDR 0) individuals.

Alzheimer's disease (AD), the main cause of dementia worldwide, is characterized by a complex and multifactorial etiology. In large part, excitatory neurotransmission in the central nervous system is mediated by glutamate and its receptors are involved in synaptic plasticity. The N-methyl-D-aspartate (NMDA) receptors, which require the agonist glutamate and a coagonist such as glycine or the D-enantiomer of serine for activation, play a main role here. A second D-amino acid, D-aspartate, acts as agonist of NMDA receptors. D-amino acids, present in low amounts in nature and long considered to be of bacterial origin, have distinctive functions in mammals. In recent years, alterations in physiological levels of various D-amino acids have been linked to various pathological states, ranging from chronic kidney disease to neurological disorders. Actually, the level of NMDA receptor signaling must be balanced to promote neuronal survival and prevent neurodegeneration: this signaling in AD is affected mainly by glutamate availability and modulation of the receptor's functions. Here, we report the experimental findings linking D-serine and D-aspartate, through NMDA receptor modulation, to AD and cognitive functions. Interestingly, AD progression has been also associated with the enzymes related to D-amino acid metabolism as well as with glucose and serine metabolism. Furthermore, the D-serine and D-/total serine ratio in serum have been recently proposed as biomarkers of AD progression. A greater understanding of the role of D-amino acids in excitotoxicity related to the pathogenesis of AD will facilitate novel therapeutic treatments to cure the disease and improve life expectancy.

L-serine is a nonessential amino acid in eukaryotic cells, used for protein synthesis and in producing phosphoglycerides, glycerides, sphingolipids, phosphatidylserine, and methylenetetrahydrofolate. Moreover, L-serine is the precursor of two relevant coagonists of NMDA receptors: glycine (through the enzyme serine hydroxymethyltransferase), which preferentially acts on extrasynaptic receptors and D-serine (through the enzyme serine racemase), dominant at synaptic receptors. The cytosolic "phosphorylated pathway" regulates de novo biosynthesis of L-serine, employing 3-phosphoglycerate generated by glycolysis and the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase (the latter representing the irreversible step). In the human brain, L-serine is primarily found in glial cells and is supplied to neurons for D-serine synthesis. Serine-deficient patients show severe neurological symptoms, including congenital microcephaly, psychomotor retardation, and intractable seizures, thus highlighting the relevance of de novo production of this amino acid in brain development and morphogenesis. Indeed, the phosphorylated pathway is strictly linked to cancer. Moreover, L-serine has been suggested as a ready-to-use treatment, as also recently proposed for Alzheimer's disease. Here, we present our current state of knowledge concerning the three mammalian enzymes of the phosphorylated pathway and known mutations related to pathological conditions: although the structure of these enzymes has been solved, how enzyme activity is regulated remains largely unknown. We believe that an in-depth investigation of these enzymes is crucial to identify the molecular mechanisms involved in modulating concentrations of the serine enantiomers and for studying the interplay between glial and neuronal cells and also to determine the most suitable therapeutic approach for various diseases.

D-amino acids research field has recently gained an increased interest since these atypical molecules have been discovered to play a plethora of different roles. In the mammalian central nervous system, d-aspartate (D-Asp) is critically involved in the regulation of glutamatergic neurotransmission by acting as an agonist of NMDA receptor. Accordingly, alterations in its metabolism have been related to different pathologies. D-Asp shows a peculiar temporal pattern of emergence during ontogenesis and soon after birth its brain levels are strictly regulated by the catabolic enzyme d-aspartate oxidase (DASPO), a FAD-dependent oxidase. Rodents have been widely used as in vivo models for deciphering molecular mechanisms and for testing novel therapeutic targets and drugs, but human targets can significantly differ. Based on these considerations, here we investigated the structural and functional properties of the mouse DASPO, in particular kinetic properties, ligand and flavin binding, oligomerization state and protein stability. We compared the obtained findings with those of the human enzyme (80% sequence identity) highlighting a different oligomeric state and a lower activity for the mouse DASPO, which apoprotein species exists in solution in two forms differing in FAD affinity. The features that distinguish mouse and human DASPO suggest that this flavoenzyme might control in a distinct way the brain D-Asp levels in different organisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Members of the T2 extracellular ribonucleases family have long been reported as stress response proteins, often involved in host defence, in many different taxonomic groups. In particular, the human RNASET2 protein (hRNASET2) has been reported as an extracellular tumor suppressor protein, endowed with the ability to act as an "alarmin" signalling molecule following its expression and secretion in the tumor microenvironment by cancer cells and the subsequent recruitment and activation of cells belonging to the host innate immune system. Many in vitro and in vivo assays have been recently reported in support of the oncosuppressive role of hRNASET2: most of them relied on genetically engineered cell lines and the use of recombinant proteins from non-mammalian sources. In order to ensure a human-like glycosylation pattern, here we report for the first time the expression of recombinant hRNASET2 in the CHO-S cell line. We established a simple one-step chromatographic purification procedure that resulted in the production of 5 mg of endotoxin-free hRNASET2 per liter of culture, with a >95% purity degree. hRNASET2 expressed in CHO-S cells displayed a high degree of glycosylation homogeneity and a secondary structure content in agreement with that determined from the crystal structure. Indeed, recombinant hRNASET2 was active at both enzymatic and functional level, as stated by a biological activity assay. The availability of a pure, homogeneous recombinant human RNASET2 would provide a key tool to better investigate its non cell-autonomous roles in the context of cancer development and growth., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Selective DAAO inhibitors have demonstrated promising therapeutic effects in clinical studies, including clinically alleviating symptoms of schizophrenic patients and ameliorating cognitive function in Alzheimer's patients with early phase. Herein we report the synthesis and preliminary evaluation of a 11 C-labeled positron emission tomography ligand based on a DAAO inhibitor, DAO-1903 (8). 11 C-Isotopologue of 8 was prepared in high radiochemical yield with high radiochemical purity (>99%) and high molar activity (>37 GBq/µmol). In vitro autoradiography studies indicated that the ligand possessed high in vitro specific binding to DAAO, while in vivo dynamic PET studies demonstrated that [ 11 C]8 failed to cross the blood-brain barrier possibly due to moderate brain efflux mechanism. Further chemical scaffold optimization is necessary to overcome limited brain permeability and improve specific binding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In some environments, a number of crops, notably maize and nuts can be contaminated by aflatoxin B 1 and related compounds resulting from the growth of aflatoxin-producing Aspergilli. Fungal peroxidases have been shown to degrade a number of mycotoxins, including aflatoxin B 1 (AFB 1 ). Therefore, the purpose of this study was to investigate the in vitro enzymatic degradation AFB 1 by a recombinant type B dye decolorizing peroxidase (Rh_DypB). Analysis of the reaction products by HPLC-MS analysis showed that under optimized conditions AFB 1 was efficiently transformed by Rh_DypB, reaching a maximum of 96% conversion after 4 days of reaction at 25 °C. Based on high resolution mass spectrometry analysis, AFB 1 was demonstrated to be quantitatively converted to AFQ 1 , a compound with a significantly lower toxicity. A number of low molecular mass compounds were also present in the final reaction mixture in small quantities. The results presented in this study are promising for a possible application of the enzyme Rh_DypB for aflatoxin reduction in feed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

α-Amino acids are present in two opposite configurations due to the presence of a central carbon atom which is a chiral center. While L-amino acids are present in large amount in nature, only tiny quantities of their D-enantiomers exist. For a long time, D-amino acids have been considered of bacterial origin only, but recently we realized that they are present in all living organisms: notably, D-amino acids play specific and relevant functions in the different organisms. Detection and quantification of D-amino acids are mandatory to shed light on their physiological roles, especially related to foods and human health. Chromatographic techniques are among the most commonly used analytical methods for the enantioseparation of amino acids. Here, we revised the latest improvements in chromatographic direct methodologies based on chiral selectors and aimed to improve analytical speed, sensitivity, robustness, and reproducibility. While current methods are well suited for D-amino acid analysis in foodstuffs and pharmaceuticals, further improvements seem required for their simultaneous, fast and sensitive detection in biological fluids, an emerging field since D-amino acids have been proposed as biomarkers of different and relevant human pathologic states.

D-enantiomers of amino acids (D-AAs) are only present in low amounts in nature, frequently at trace levels, and for this reason, their biological function was undervalued for a long time. In the past 25 years, the improvements in analytical methods, such as gas chromatography, HPLC, and capillary electrophoresis, allowed to detect D-AAs in foodstuffs and biological samples and to attribute them specific biological functions in mammals. These methods are time-consuming, expensive, and not suitable for online application; however, life science investigations and industrial applications require rapid and selective determination of D-AAs, as only biosensors can offer. In the present review, we provide a status update concerning biosensors for detecting and quantifying D-AAs and their applications for safety and quality of foods, human health, and neurological research. The review reports the main challenges in the field, such as selectivity, in order to distinguish the different D-AAs present in a solution, the simultaneous assay of both L- and D-AAs, the production of implantable devices, and surface-scanning biosensors. These innovative tools will push future research aimed at investigating the neurological role of D-AAs, a vibrant field that is growing at an accelerating pace., Competing Interests: The authors declare no conflict of interest.

pLG72 is a primate-specific protein of enigmatic function that was proposed to modulate mitochondria fragmentation and the activity of the peroxisomal enzyme D-amino acid oxidase (DAAO). DAAO is deputed to degradation of the NMDA receptor co-agonist D-serine in human brain and the R199W substitution in DAAO was identified in a familial case of amyotrophic lateral sclerosis (ALS). A recent work reported that U87 glioblastoma cells ectopically expressing pLG72 showed a lower proliferation, produced superoxide radicals, induced SOD1 aggregation and decreased its activity. Because of the role of SOD1 in eliminating ROS species and its relevance in ALS we evaluated the link between pLG72 and SOD1 using both wild-type pLG72 and its R30K variant related to schizophrenia susceptibility. In vitro studies on recombinant proteins excluded the establishment of a stable complex and that pLG72 could affect SOD1 activity and stability. At cellular level, ectopic expression of pLG72 in glioblastoma U87 cells did not affect cell viability and ROS/superoxide production: only caspase activity (a marker of apoptosis) was slightly increased in cells expressing the R30K pLG72 variant. SOD1 and pLG72 did not colocalize in transfected U87 glioblastoma cells: pLG72 largely localised to mitochondria and SOD1 was largely cytosolic. Moreover, the ectopic expression of pLG72 appeared not to alter the expression of SOD1 and its aggregation. Altogether, the combination of biochemical and cellular studies allow to exclude that pLG72 modulates SOD1 function and aggregation, thus that it could play a role in ALS susceptibility.

In nature, the D-enantiomers of amino acids (D-AAs) are not used for protein synthesis and during evolution acquired specific and relevant physiological functions in different organisms. This is the reason for the surge in interest and investigations on these "unnatural" molecules observed in recent years. D-AAs are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. In past years, a number of methods have been devised to produce D-AAs based on enantioselective enzymes. With the aim to increase the D-AA derivatives generated, to improve the intrinsic atomic economy and cost-effectiveness, and to generate processes at low environmental impact, recent studies focused on identification, engineering and application of enzymes in novel biocatalytic processes. The aim of this review is to report the advances in synthesis of D-AAs gathered in the past few years based on five main classes of enzymes. These enzymes have been combined and thus applied to multi-enzymatic processes representing in vitro pathways of alternative/exchangeable enzymes that allow the generation of an artificial metabolism for D-AAs synthetic purposes.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein ( Hv RNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, Hv RNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of Hv RNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. Hv RNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released Hv RNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area., (Copyright © 2020 Baranzini, De Vito, Orlandi, Reguzzoni, Monti, de Eguileor, Rosini, Pollegioni, Tettamanti, Acquati and Grimaldi.)

The flavoenzyme D-amino acid oxidase (DAAO) represents a potentially good option for cancer enzyme prodrug therapy as it produces H 2 O 2 using D-amino acids as substrates, compounds present at low concentration in vivo and that can be safely administered to regulate H 2 O 2 production. We optimized the cytotoxicity of the treatment by: i) using an efficient enzyme variant active at low O 2 and D-alanine concentrations (mDAAO); ii) improving the stability and half-life of mDAAO and the enhanced permeability and retention effect by PEGylation; and iii) inhibiting the antioxidant cellular system by a heme oxygenase-1 inhibitor (ZnPP). A very low amount of PEG-mDAAO (10 mU, 50 ng of enzyme) induces cytotoxicity on various tumor cell lines. Notably, PEG-mDAAO seems well suited for in vivo evaluation as it shows the same cytotoxicity at air saturation (21%) and 2.5% O 2 , a condition resembling the microenvironment found in the central part of tumors., (Copyright © 2019 Elsevier Inc. All rights reserved.)

d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-d-aspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first three-dimensional structure of a DASPO enzyme (from human) which belongs to the d-amino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR) showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target., (© 2019 Federation of American Societies for Experimental Biology.)

With the only exception of glycine, all amino acids exist in two specular structures which are mirror images of each other, called D-(dextro) and L-(levo) enantiomers. During evolution, L-amino acids were preferred for protein synthesis and main metabolism; however, the D-amino acids (D-AAs) acquired different and specific functions in different organisms (from playing a structural role in the peptidoglycan of the bacterial cell wall to modulating neurotransmission in mammalian brain). With the advent of sophisticated and sensitive analytical techniques, it was established during the past few decades that many foods contain considerable amounts of D-AAs: we consume more than 100 mg of D-AAs every day. D-AAs are present in a variety of foodstuffs, where they fulfill a relevant role in producing differences in taste and flavor and in their antimicrobial and antiaging properties from the corresponding L-enantiomers. In this review, we report on the derivation of D-AAs in foods, mainly originating from the starting materials, fermentation processes, racemization during food processing, or contamination. We then focus on leading-edge methods to identify and quantify D-AAs in foods. Finally, current knowledge concerning the effect of D-AAs on the nutritional state and human health is summarized, highlighting some positive and negative effects. Notwithstanding recent progress in D-AA research, the relationships between presence and nutritional value of D-AAs in foods represent a main scientific issue with interesting economic impact in the near future.

Among nanocelluloses, bacterial nanocellulose (BNC) has proven to be a promising candidate in a range of biomedical applications, from topical wound dressings to tissue-engineering scaffolds. Chemical modifications and incorporation of bioactive molecules have been obtained, further increasing the potential of BNC. This study describes the incorporation of vancomycin and ciprofloxacin in BNC and in modified BNC to afford bioactive BNCs suitable for topical wound dressings and tissue-engineering scaffolds. BNC was modified by grafting glycidylmethacrylate (GMA) and further cross-linking with ethylene glycol dimethacrylate (EGDMA) with the formation of stable C-C bonds though a radical Fenton-type process that involves generation of cellulose carbon centred radicals scavenged by methacrylate structures. The average molar substitution degree MS (MS = methacrylate residue per glucose unit, measured by Fourier transform infrared (FT-IR) analysis) can be modulated in a large range from 0.1 up to 3. BNC-GMA, BNC-EGDMA and BNC-GMA-EGDMA maintain the hydrogel status until MS reaches the value of 1. The mechanical stress resistance increase of BNC-GMA and BNC-GMA-EGDMA of MS around 0.8 with respect to BNC suggests that they can be preferred to BNC for tissue-engineering scaffolds in cases where the resistance plays a crucial role. BNC, BNC-GMA, BNC-EGDMA and BNC-GMA-EGDMA were loaded with vancomycin (VC) and ciprofloxacin (CP) and submitted to release experiments. BNC-GMA-EGDMA of high substitution degree (0.7-1) hold up to 50 percentage of the loaded vancomycin and ciprofloxacin amount, suggesting that they can be further investigated for long-term antimicrobial activity. Furthermore, they were not colonized by Staphylococcus aureus (S.A.) and Klebsiella pneumonia (K.P.). Grafting and cross-linking BNC modification emerges from our results as a good choice to improve the BNC potential in biomedical applications like topical wound dressings and tissue-engineering scaffolds., Competing Interests: The authors declare no conflict of interest.

The peroxisomal enzyme human D-amino acid oxidase (hDAAO) is attracting attention owing to its role in degrading D-serine, the main co-agonist of N-methyl D-aspartate receptors in brain, and its involvement in brain functions and diseases. Here, we focused on arginine 120, a residue located at the protein interface, 20 Å from the assumed second ligand-binding site, showing a different orientation of the side chain in the hDAAO-benzoate complex, and corresponding to Ser119 in rat DAAO, which is part of a putative nuclear translocation signal (NTS). By substituting Arg120 in hDAAO with a glutamate (to mimic the active NTS) or a leucine (to eliminate the positive charge) the protein conformation, thermal stability, and kinetic properties are slightly altered, while the dimeric structure and the ligand-binding properties are unchanged. The most relevant alteration in Arg120 variants is the strongest interaction with FAD. Nevertheless, the activity assayed at low D-serine and FAD concentrations (resembling physiological conditions) was quite similar for wild-type and Arg120 hDAAO variants. These results resemble the ones obtained substituting another residue located at the interface region (i.e., the W209R variant), indicating that substitutions at the monomer-monomer interface mainly affects the FAD binding in hDAAO. Indeed, U87 glioblastoma cells transiently transfected for hDAAO variants show that substitution of Arg120 favors mistargeting: the increase in cytosolic localization observed for the variants promotes nuclear targeting, especially for the R120E hDAAO, without affecting cell viability. Notably, mistargeting to the nucleus is an innate process as it is apparent for the wild-type hDAAO, too: whether such a process is related to specific pathologic processes is still unknown., (Copyright © 2019 Murtas, Sacchi and Pollegioni.)

Protein engineering represents a modern approach to generate novel proteins for the different fields of biotechnology. Here, we report about an 8-day laboratory activity in which students generate enzyme variants to degrade the herbicide glyphosate. The students conduct a true research experiment in an important field (bioremediation by novel, engineered enzymes) and are introduced to widely used techniques in molecular biology and protein biochemistry laboratories. Based on a docking analysis of glycine (the original substrate) and of glyphosate into the active site of glycine oxidase, residues putatively involved in substrate selectivity are identified that will become the target of site-saturation mutagenesis. Each group of students focuses on the library generated at one position and selects the most active variant based on colorimetric screening. Following protein overexpression in Escherichia coli, the selected glycine oxidase variants are purified and their kinetic properties on glycine and glyphosate assessed. The best variant identified by the whole class is then used for detecting the herbicide in water. With the help of the professor, students can improve technical skills, ability to evaluate results, team work activity, and critical thinking. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):689-699, 2019., (© 2019 International Union of Biochemistry and Molecular Biology.)