The cAMP-dependent phosphorylation footprint in response to heat stress.

KEY MESSAGE: cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP-dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular "sponge" that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

On the regulation of human D-aspartate oxidase.

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 H2 O2 produced by the hDASPO enzymatic degradation of D-aspartate, especially before the final targeting to peroxisomes.

Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives.

The accumulation of synthetic plastic waste in the environment has become a global concern. Microbial enzymes (purified or as whole-cell biocatalysts) represent emerging biotechnological tools for waste circularity; they can depolymerize materials into reusable building blocks, but their contribution must be considered within the context of present waste management practices. This review reports on the prospective of biotechnological tools for plastic bio-recycling within the framework of plastic waste management in Europe. Available biotechnology tools can support polyethylene terephthalate (PET) recycling. However, PET represents only ≈7% of unrecycled plastic waste. Polyurethanes, the principal unrecycled waste fraction, together with other thermosets and more recalcitrant thermoplastics (e.g., polyolefins) are the next plausible target for enzyme-based depolymerization, even if this process is currently effective only on ideal polyester-based polymers. To extend the contribution of biotechnology to plastic circularity, optimization of collection and sorting systems should be considered to feed chemoenzymatic technologies for the treatment of more recalcitrant and mixed polymers. In addition, new bio-based technologies with a lower environmental impact in comparison with the present approaches should be developed to depolymerize (available or new) plastic materials, that should be designed for the required durability and for being susceptible to the action of enzymes.

Efficient polyethylene terephthalate degradation at moderate temperature: a protein engineering study of LC-cutinase highlights the key role of residue 243.

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.

Preliminary investigation of microorganisms potentially involved in microplastics degradation using an integrated metagenomic and biochemical approach.

Plastic pollution is becoming an emerging environmental issue due to inappropriate disposal at the end of the materials life cycle. When plastics are released, they undergo physical and chemical corrosion, leading to the formation of small particles, commonly referred to as microplastics. In this study, a microbial community derived from the leachate of a bioreactor containing a mixture of soil and plastic collected during a landfill mining process underwent an enrichment protocol in order to select the microbial species specifically involved in plastic degradation. The procedure was set up and tested on polyethylene, polyvinyl chloride, and polyethylene terephthalate, both in anaerobic and aerobic conditions. The evolution of the microbiome has been monitored using a combined approach based on microscopy, marker-gene amplicon sequencing, genome-centric metagenomics, degradation assays, and GC-MS analyses. This procedure permitted us to deeply investigate the metabolic pathways potentially involved in plastic degradation and to depict the route for microplastics metabolization from the enriched microbial community. Six enzymes, among the ones already identified, were found in our samples (alkane 1-monooxygenase, cutinase, feruloyl esterase, triacylglycerol lipase, medium-chain acyl-CoA dehydrogenase, and protocatechuate 4,5-dioxygenase) and new enzymes, addressed as MHETases most probably for the presence of the catalytic triad (His-Asp-Ser), were detected. Among the enzymes involved in plastics degradation, alkane 1-monooxygenase was found in high copy number (between ten and 62 copies) in the metagenomes that resulted most abundant in the microbiome enriched with polyethylene, while protocatechuate 4,5-dioxygenase was found between one and eight copies in the most abundant metagenomes of the microbial culture enriched with polyethylene terephthalate. Degradation assays, performed using both bacterial lysates and supernatants, revealed interesting results on polyethylene terephthalate degradation. Moreover, this study demonstrates to what extent different types of microplastics can affect the microbial community composition. The results obtained significantly increase the knowledge of the plastic degradation process.

Biochemical Properties and Physiological Functions of pLG72: Twenty Years of Investigations.

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.

The conundrum in enzymatic reactions related to biosynthesis of d-amino acids in bacteria.

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.

Human D-aspartate Oxidase: A Key Player in D-aspartate Metabolism.

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.

Analytical methods for the investigation of enzyme-catalyzed degradation of polyethylene terephthalate.

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.

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes.

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.

Succinic Semialdehyde Dehydrogenase Deficiency: In Vitro and In Silico Characterization of a Novel Pathogenic Missense Variant and Analysis of the Mutational Spectrum of ALDH5A1.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter γ-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including γ-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.

Biochemical characterization of mouse d-aspartate oxidase.

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.

Advances in Enzymatic Synthesis of D-Amino Acids.

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 Protein Imager: a full-featured online molecular viewer interface with server-side HQ-rendering capabilities.

SUMMARY: Molecular viewers' long learning curve is hindering researchers in approaching the field of structural biology for the first time. Herein, we present 'The Protein Imager', a lightweight, powerful and easy-to-use interface as a next-gen online molecular viewer. Furthermore, the interface is linked to an automated server-side rendering system able to generate publication-quality molecular illustrations. The Protein Imager interface has been designed for easy usage for beginners and experts in the field alike. The interface allows the preparation of very complex molecular views maintaining a high level of responsiveness even on mobile devices. AVAILABILITY AND IMPLEMENTATION: The Protein Imager interface is freely available online at https://3dproteinimaging.com/protein-imager. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain.

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.

Properties of l-amino acid deaminase: En route to optimize bioconversion reactions.

Interest is rising in the agrochemical and pharmaceutical industries concerning the use of enantiomerically pure amino acids. l-Amino acids are easily produced by deracemization of D,L-mixtures or by stereoinversion of d-amino acids, employing the flavoenzyme d-amino acid oxidase. On the other hand, the production of the D-enantiomers is hampered by the lack of a suitable enzyme with reversed stereoselectivity. In recent years, the enzyme l-amino acid deaminase has been proposed as an alternative to l-amino acid oxidase. l-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a membrane-bound flavoprotein that catalyzes the deamination of l-amino acids to the corresponding α-keto acids and ammonia without producing hydrogen peroxide since the electrons are transferred from the reduced cofactor to a b-type cytochrome. For this reason, purified PmaLAAD has no significant enzymatic activity; this can be recovered by adding exogenous E. coli membranes. In order to circumvent the use of membranes, we analyzed the ability of PmaLAAD to use alternative electron acceptors, as well as detergents, to reproduce the hydrophobic environment. With phenazine methosulfate (PMS) and anionic detergents, at concentrations lower than the critical micellar concentration, higher enzymatic activity can be reached than with membranes. The effect on stability, protein conformation, oligomeric state and activity of temperature, pH, ionic strength, and detergents was also investigated. By optimizing the reaction conditions (namely, using 0.8 mM PMS and 0.1 mM SDS) the rate of l-leucine bioconversion was improved.

D-Amino Acid Oxidase-pLG72 Interaction and D-Serine Modulation.

pLG72 is a small, primate-specific protein of 153 amino acids. It is the product of the G72 gene, expressed in testis, spinal cord, and brain. The presence of G72 transcript and pLG72 has recurrently been called into question, however G72 mRNA and pLG72 protein levels were higher in blood and brain of patients with schizophrenia than in healthy controls. On the one hand, the SNP rs2391191 corresponding to the R30K substitution in pLG72 was genetically linked to schizophrenia, reduced thickness of the brain cortex in schizophrenia-affected individuals, and altered memory function. Various lines of evidence indicated that pLG72 is a mitochondrial protein, specifically an extrinsic protein bound on the outer membrane. Over the years, pLG72 was proposed to be involved in different functions: (a) overexpression induces mitochondria fragmentation, increasing the numbers of shorter and more mobile ones which could be delivered faster to regions of intense growth and facilitating the dendritic complexity; (b) it might induce oxidative stress by interacting with methionine-R-sulfoxide reductase B2; and (c) it binds and modulates the activity of FMN-containing oxidoreductase of the respiratory complex I. The main role of this protein, however, is related to its binding to the human flavoenzyme D-amino acid oxidase (hDAAO), i.e., the main catabolic enzyme for D-enantiomer of serine. This D-amino acid is a main endogenous coagonist of the N-methyl-D-aspartate type glutamate receptor (NMDAR) involved in main functions such as synaptic plasticity, learning, memory, and excitotoxicity. For this work, we reviewed the recent literature concerning the hDAAO-pLG72 interaction, focusing on the molecular details of the interaction, the effect of hDAAO function and stability, and the cellular effects, especially on D-serine concentration. The main effects related to the pathological R30K substitution are also reported. We have highlighted the gaps in our knowledge of this human protein as well as the relevance of clarifying the molecular details of hDAAO-pLG72 interaction in order to design molecules to modulate hDAAO activity/stability and thus NMDAR function acting at the D-serine cellular level.

Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design.

An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.