Is rat an appropriate animal model to study the involvement of d-serine catabolism in schizophrenia? insights from characterization of d-amino acid oxidase.

d-Amino acid oxidase (DAAO; EC1.4.3.3) has been proposed to play a main role in the degradation of d-serine, an allosteric activator of the N-methyl-d-aspartate-type glutamate receptor in the human brain, and to be associated with the onset of schizophrenia. To prevent excessive d-serine degradation, novel drugs for schizophrenia treatment based on DAAO inhibition were designed and tested on rats. However, the properties of rat DAAO are unknown and various in\u2003vivo trials have demonstrated the effects of DAAO inhibitors on d-serine concentration in rats. In the present study, rat DAAO was efficiently expressed in Escherichia\u2003coli. The recombinant enzyme was purified as an active, 40\u2003kDa monomeric flavoenzyme showing the basic properties of the dehydrogenase-oxidase class of flavoproteins. Rat DAAO differs significantly from the human counterpart because: (a) it possesses a different substrate specificity; (b) it shows a lower kinetic efficiency, mainly as a result of a low substrate affinity; (c) it differs in affinity for the binding of classical inhibitors; (d) it is a stable monomer in the absence of an active site ligand; and (e) it interacts with the mammalian protein modulator pLG72 yielding a 3c\u2003100\u2003kDa complex in addition to the 3c\u2003200\u2003kDa one, as formed by the human DAAO. Furthermore, the concentration of endogenous d-serine in U87 glioblastoma cells was not affected by transfection with rat DAAO, whereas it was significantly decreased when expressing the human homologue. These results raise doubt on the use of the rat as a model system for testing new drugs against schizophrenia and indicate a different physiological function of DAAO in rodents and humans. Structured digital abstract \u2022 \u2002pLG72\ua0binds\u2003rDAAO\u2003by\u2003molecular sieving\u2003(View interaction)

ONE-POT ENZIMATIC DEPOLYMERIZATION OF CELLULOSE IN IONIC LIQUIDS

Green alternatives to fossil-based fuels are very attractive and can be produced from cellulosic materials. Cellulose is the primary product of photosynthesis in plants and has immense importance as a renewable raw material. The production of biofuels starting from cellulose is gaining increasing attention and obviously implies the partial or total hydrolysis of cellulose: enzymatic processes are considered the most promising technology [1]. Cellulases (EC 3.2.1.4) are the enzymes most commonly employed to selectively depolymerize cellulose in buffered aqueous solvents. Because of the very low solubility of cellulose due to its highly organized structure, enzymatic conversions proceed at very slow reaction rates and require the dissolution in a solvent to facilitate the access of cellulases to cellulosic substrates. To improve the yield of fermentable monosaccharides, pretreatments of cellulose, such as thermal, chemical or physical treatment, have been applied to afford a better enzymatic conversion [2]. Ionic liquids (ILs) have been increasingly recognized as excellent solvents for dissolution and pretreatment of cellulose but it was previously reported that ILs induce usually fast enzyme deactivation by protein unfolding [3]. In the present work we present a study on a single-batch, homogeneous phase enzymatic hydrolysis of cellulose using three commercial ILs. We have tested two native proteins from Trichoderma reesei and Humicola insolens and two engineered proteins from T. reesei and Streptomyces sp.. In some cases ILs don’t denature the cellulases used but increase their operational stability as compared to standard buffer solutions and facilitate the dissolution of cellulose. Interestingly, the stability of the four cellulases in the presence of the ILs allows to set-up a procedure lacking of the cellulose pretreatment step. We believe that this strategy could be amenable of scale-up and innovative industrial applications for the efficient one-batch conversion of inexpensive cellulosic materials into derivatives (biofuels, derivatized cellulose, monosaccharides for fine chemicals, etc.) with high potential commercial interest and in the framework of environmentally friendly chemistry. References [1] A.P. dadi, S. Varanasi, C.A. Schall. Biotechnol Bioeng, 95(5), 904-910, (2006). [2] M.B. Turner, S.K. Spear, J.G. Huddleston, J.D. Holbrey, R.D. Rogers. Green Chem, 5(4), 443-447, (2003). [3] S.D. Zhu, Y.X. Wu, Q.M. Chen, C. Wang, S. Jin, Y. Ding, G. Wu, Green Chem, 8, 325-327, (2006)

Optimizing HIV-1 protease production in Escherichia coli as fusion protein

<p>Abstract</p> <p>Background</p> <p>Human immunodeficiency virus (HIV) is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the <it>pol </it>gene is responsible for proteolytic processing of the <it>gag-pol </it>polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr) is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations.</p> <p>Results</p> <p>A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr) was overexpressed in <it>Escherichia coli </it>cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA) or glutathione S-transferase (GST), also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for <it>E. coli </it>expression and to avoid autoproteolysis) and by screening six different <it>E. coli </it>strains and five growth media. The best expression yields were achieved in <it>E. coli </it>BL21-Codon Plus(DE3)-RIL host and in TB or M9 medium to which 1% (w/v) glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts) and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth). GST:HIVPr was in part (50%) produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1Pr per liter.</p> <p>Conclusions</p> <p>By using this optimized expression and purification procedure fairly large amounts of good-quality HIV-1Pr recombinant enzyme can be produced at the lab-scale and thus used for further biochemical studies.</p

Whole-Cell Bioconversion of Renewable Biomasses-Related Aromatics to cis,cis-Muconic Acid

Lignin and wheat bran represent renewable feedstocks for generation of useful and value-added compounds such as vanillin (a popular flavoring agent) and cis,cis-muconic acid (ccMA, a building block for the synthesis of plastic materials). In the present work, we report on the setup of an efficient and green process for producing such valuable compounds based on (a) the optimization of the extraction procedures for vanillin from lignin and ferulic acid from wheat bran and (b) the genetic engineering of an Escherichia coli strain with up to three plasmids differing in copy numbers to modulate the expression of up to seven recombinant enzymes. In detail, we used two sequential reactions catalyzed by the decarboxylase Fdc and the dioxygenase Ado to convert wheat bran-derived ferulic acid into vanillin: nature-identical vanillin was produced in one pot with a &gt;85% yield in 20 h. Next, the dehydrogenase LigV, the demethylase VanAB, the decarboxylase AroY, and the dioxygenase C12O converted lignin-derived vanillin into ccMA with a &gt;95% conversion yield and a productivity of 4.2 mg of ccMA/g of Kraft lignin in 30 min. Finally, when the optimized E. coli strain expressing all the abovementioned enzymes was used, ccMA was produced with a &gt;95% conversion yield starting from ferulic acid in 10 h following product isolation, corresponding to 0.73 g of ccMA/g of ferulic acid, 1.4 g of ccMA/L, and 2.2 g of ccMA/g of wheat bran biomass. The optimized whole-cell system represents a sustainable and cost-competitive process for producing high value-added products from renewable resources

The symmetric active site of enantiospecific enzymes

Biomolecules are frequently chiral compounds, existing in enantiomeric forms. Amino acids represent a meaningful example of chiral biological molecules. Both L- and D-amino acids play key roles in the biochemical structure and metabolic processes of living organisms, from bacteria to mammals. In this review, we explore the enantiospecific interaction between proteins and chiral amino acids, introducing theoretical models and describing the molecular basis of the ability of some of the most important enzymes involved in the metabolism of amino acids (i.e., amino acid oxidases, dehydrogenases, and aminotransferases) to discriminate the opposite enantiomers. Our analysis showcases the power of natural evolution in shaping biological processes. Accordingly, the importance of amino acids spurred nature to evolve strictly enantioselective enzymes both through divergent evolution, starting from a common ancestral protein, or through convergent evolution, starting from different scaffolds: intriguingly, the active sites of these enzymes are frequently related by a mirror symmetry

Evidence for the interaction of d-amino acid oxidase with pLG72 in a glial cell line.

Accumulating genetic evidence indicates that the primate-specific gene locus G72/G30 is related to schizophrenia: it encodes for the protein pLG72, whose function is still the subject of controversy. We recently demonstrated that pLG72 negatively affects the activity of human d-amino acid oxidase (hDAAO, also related to schizophrenia susceptibility), which in neurons and (predominantly) in glia is expected to catabolize the neuromodulator d-serine. The d-serine regulation mechanism relying on hDAAO-pLG72 interaction does not match with the subcellular localizations proposed for hDAAO (peroxisomes) and pLG72 (mitochondria). By using glioblastoma U87 cells transfected with plasmids encoding for hDAAO and/or pLG72 we provide convergent lines of evidence that newly synthesized hDAAO, transitorily present in cytosol before being delivered to the peroxisomes, colocalizes and interacts with pLG72 which we propose to be exposed on the external membrane of mitochondria. We also report that newly synthesized cytosolic hDAAO is catalytically active, and therefore pLG72 binding-and ensuing hDAAO inactivation-plays a protective role against d-serine depletion

Lignin valorization: production of high value-added compounds by engineered microorganisms

Lignin is the second most abundant polymer in nature, which is also widely generated during biomass fractionation in lignocellulose biorefineries. At present, most of technical lignin is simply burnt for energy supply although it represents the richest natural source of aromatics, and thus it is a promising feedstock for generation of value-added compounds. Lignin is heterogeneous in composition and recalcitrant to degradation, with this substantially hampering its use. Notably, microbes have evolved particular enzymes and specialized metabolic pathways to degrade this polymer and metabolize its various aromatic components. In recent years, novel pathways have been designed allowing to establish engineered microbial cell factories able to efficiently funnel the lignin degradation products into few metabolic intermediates, representing suitable starting points for the synthesis of a variety of valuable molecules. This review focuses on recent success cases (at the laboratory/pilot scale) based on systems metabolic engineering studies aimed at generating value-added and specialty chemicals, with much emphasis on the production of cis,cis-muconic acid, a building block of recognized industrial value for the synthesis of plastic materials. The upgrade of this global waste stream promises a sustainable product portfolio, which will become an industrial reality when economic issues related to process scale up will be tackled

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

Characterization of the human D-amino acid oxidase (hDAAO) - pLG72 complex involved in the onset of schizophrenia.

Schizophrenia is a chronic and severely debilitating psychiatric disorder affecting nearly 1% of the world’s population. In 2002, the new human gene G72, encoding for the pLG72 protein, and the gene encoding for DAAO have been genetically linked to the susceptibility to schizophrenia. A yeast two-hybrid screening experiment identified D-amino acid oxidase (DAAO) as a putative interacting partner of pLG72. DAAO is a FAD-containing flavooxidase that in brain is responsible for the elimination of D-serine, a co-agonist that binds to the glycine-site of the NMDA receptor. We recently demonstrated that pLG72 acts as “inactivator” of human DAAO and that the cellular concentration of D-serine depends on the expression of the active form of this flavooxidase. Based on these results, a molecular model for the onset of schizophrenia has been proposed: a decrease in pLG72 expression might yield an anomalous high level of hDAAO activity and therefore a decrease in the local concentration of D-serine, affecting glutamatergic neurotransmission mediated by NMDA receptor. The characterization of the complex is a challenging task, hardly feasible by high resolution techniques, since: 1) structural informations on pLG72 are lacking; 2) pLG72 is soluble only in the presence of mild denaturant; 3) no homologous protein has been structurally characterized so far. In this perspective, we have used low resolution strategies based on the coupling of classical biochemistry approaches (complementary proteolysis, cross-link) with mass spectrometric techniques, to characterize the pLG72-hDAAO complex. Results indicated that hDAAO exhibits different proteolysis profiles when isolated or in complex with pLG72, thus suggesting a conformational change upon binding the effector protein. Chemical cross-linking experiments will complement the proteolysis experiments providing with details the contact regions between hDAAO e pLG72