One single method to produce native and Tat-fused recombinant human \u3b1-synuclein in Escherichia coli

Human \u3b1-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that \u3b1-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of \u3b1-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate \u3b1-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-\u3b1-synuclein.A reliable protocol was designed to efficiently express and purify two different forms of human \u3b1-synuclein. The synthetic cDNAs encoding for the native \u3b1-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized ( 65 85\%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant \u3b1-synuclein protein forms could be purified in a single step to 65 95\% purity. Both \u3b1-synuclein recombinant proteins form fibrils and the TAT-\u3b1-synuclein is also cytotoxic in the micromolar concentration range.To further characterize the molecular mechanisms of \u3b1-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular \u3b1-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant \u3b1-synuclein than previously described procedures

Assays of D-amino acid oxidase activity

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or \u3b1-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the \u3b1-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples

Role of human DAAO in D-serine metabolism: new insight

D-amino acid oxidase (DAAO) is a peroxisomal flavoenzyme significantly enriched in the mammalian brain. It has been proposed to play (with serine racemase) an essential role in the catabolism of the \u201catypical\u201d transmitter-like molecule D-serine, an allosteric activator of the NMDA receptor (NMDAr). Indeed, D-serine cellular concentration depends on the expression of active DAAO. Alterations in the enzyme functionality and/or in its cellular levels might contribute to D-serine signaling dysregulation and the associated NMDAr dysfunctions that occur in several pathological conditions, including neurodegenerative diseases and psychiatric disorders. Noteworthy, genetic evidences indicate that human DAAO (hDAAO) - and its negative regulator pLG72 \u2013 are related to schizophrenia. We demonstrated that newly synthesized hDAAO is active and interacts with its modulator in the cytosol being progressively inactivated. The largest part of hDAAO (a long lived protein) is degraded by the lysosomal system, while pLG72 (showing a rapid turnover) is mainly targeted to the proteasome: pLG72 binding destabilizes hDAAO and increases its degradation, likely playing a protective role against excessive D-serine depletion. Furthermore, we investigated the effect of SNPs in hDAAO potentially related to schizophrenia. D31H and R279A variants show an increased activity and their expression in U87 cells produced an higher decrease in D-serine cellular level than the wild-type counterpart. These substitutions could negatively affect the concentration in vivo of the neuromodulator and thus might be relevant for schizophrenia susceptibility

Conversion of the dimeric D-amino acid oxidase from Rhodotorula gracilis to a monomeric form. A rational mutagenesis approach

AbstractThe relevance of the dimeric state for the structure/function relationships of Rhodotorula gracilis D-amino acid oxidase (RgDAAO) holoenzyme has been investigated by rational mutagenesis. Deletion of 14 amino acids in a surface loop (connecting β-strands 12 and 13) transforms RgDAAO from a dimeric protein into a stable monomer. The mutant enzyme is still catalytically competent and retains its binding with the FAD coenzyme. Dimerization has been used by this flavoenzyme in evolution to achieve maximal activity, a tighter interaction between the protein moiety and the coenzyme, and higher thermal stability

Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: A comparison between proteins from different sources

Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, d-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and d-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs.Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, D-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and D-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs