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|Autori: ||Frattini, Luca Franco|
|Tutor interno: ||PIUBELLI, LUCIANO|
|Titolo: ||Amino acid oxidases in red biotechnologies: a target and a tool.|
|Abstract: ||D-Amino acid oxidase (DAAO; EC 126.96.36.199) 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, 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. The properties of rat DAAO (rDAAO) are unknown and various in vivo trials reported on the effects of DAAO inhibitors on D-serine concentration in rats. rDAAO was efficiently expressed in Escherichia coli. The recombinant enzyme was purified as an active, 40 kDa monomeric flavoenzyme showing the basic properties of the dehydrogenase-oxidase class of flavoproteins. rDAAO differs significantly from the human enzyme because it: 1)) possesses a different substrate specificity; 2) shows a lower kinetic efficiency (because of a low substrate affinity); 3) differs in affinity for binding of classical inhibitors; 4) is a stable monomer; 5) interacts with the mammalian protein modulator pLG72 yielding a 100 kDa complex in addition to the 200 kDa, as formed by the human DAAO. Interestingly, the concentration of endogenous D-serine in U87 glioblastoma cells was not affected by transfection with rDAAO whereas it was significantly decreased when expressing the human homologue. These results raise doubt on the use of rat as model system for testing new drugs against schizophrenia and indicate a different physiological function of DAAO in rodents and humans.
During past years, a number of variants of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all D-amino acids) both by rational design and directed evolution methods have been produced in our laboratory. RgDAAO is the most suitable biotechnological tool for the detection of D-amino acids and in this work we evaluated the capability of some mutant forms of this flavoenzyme previously produced in our laboratory in order to determine D-amino acid content in different biological samples. The kinetic constants for a number of natural and unnatural D-amino acids have been investigated. This information constitutes the basis for considering potential analytical applications of these variants of RgDAAO.
Glycine is implicated in several physiological functions, e.g. as a biosynthetic precursor or neurotransmitter in the central nervous system. Glycine is an important coagonist of NMDA receptor and it is putatively involved in schizophrenia susceptibility and other neurological diseases, such as congenital nonketotic hyperglycinemia. With the final aim to produce an optimized enzyme that can be employed in a specific biosensor for glycine detection, glycine oxidase from Bacillus subtilis (GO) was engineered to improve its kinetic efficiency on this small amino acid. Based on in silico analysis, site saturation mutagenesis was independently performed at positions Met49, Gly51, Ala54, Met95, Tyr241, His244, Tyr246, Met261, Arg302, Arg329 and Asn330. The GO variants were screened by employing a rapid colorimetric assay on 96 well-plates based on the determination of hydrogen peroxide produced on glycine as substrate: seven GO variants were selected. Significant alteration of kinetic parameters was observed for H244K and H244R GO variants: kcat increased about twice and Km decreased 3-5-fold, yielding a 7-12-fold higher kinetic efficiency on glycine, as compared to the wild-type GO. Screening of GO variants at position 49 also identified an improved enzyme (M49I) showing a 1.4-fold decreased Km. Combination of information gathered from the site saturation mutagenesis approach could be useful to obtain an evolved GO variant suitable for biotechnological applications.|
|Parole chiave: ||D-Amino acid oxidases, glycine oxydase, biosensors schizophrenia, flavoxidases.|
|Corso di dottorato: ||Biologia Cellulare e Molecolare|
|Ciclo di dottorato: ||25|
|Università di conseguimento titolo: ||Università degli Studi dell'Insubria|
|Citazione: ||Frattini, L.F.Amino acid oxidases in red biotechnologies: a target and a tool. (Doctoral Thesis, Università degli Studi dell'Insubria, 2012).|
|Phd_thesis_frattinilucafranco_completa.pdf||testo completo tesi||8,56 MB||Adobe PDF||non consultabile
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