D-amino acid oxidase (DAAO) is a FAD-containing flavoprotein that dehydrogenates the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD. The cofactor then reoxidizes on molecular oxygen and the imino acid hydrolyzes spontaneously to the alpha-keto acid and ammonia. In vitro DAAO displays broad substrate specificity, acting on several neutral and basic D-amino acids: the most efficient substrates are amino acids with hydrophobic side chains. D-aspartic acid and D-glutamic acid are not substrates for DAAO. Through the years, it has been the subject of a number of structural, functional and kinetic investigations. The most recent advances are represented by site-directed mutagenesis studies and resolution of the 3D-structure of the enzymes from pig, human and yeast. The two approaches have given us a deeper understanding of the structure-function relationships and promoted a number of investigations aimed at the modulating the protein properties. By a rational and/or a directed evolution approach, DAAO variants with altered substrate specificity (e.g., active on acidic or on all D-amino acids), increased stability (e.g., stable up to 60 degrees C), modified interaction with the flavin cofactor, and altered oligomeric state were produced. The aim of this paper is to provide an overview of the most recent research on the engineering of DAAOs to illustrate their new intriguing properties, which also have enabled us to pursue new biotechnological applications.

Y238, one of the very few conserved residues in the active site of D-amino acid oxidases (DAAO), was mutated to phenylalanine and serine in the enzyme from the yeast Rhodotorula gracilis. The mutated proteins are catalytically competent thus eliminating Tyr238 as an active-site acid/ base catalyst. Y238F and Y238S mutants exhibit a threefold slower turnover on D-alanine as substrate, which can be attributed to a slower rate of product release relative to the wild-type enzyme (a change of the rate constants for substrate binding was also evident). The Y238 DAAO mutants have spectral properties similar to those of the wild-type enzyme but the degree of stabilization of the flavin semiquinone and the redox properties in the free form of Y238S are different. The binding of the carboxylic acid competitive inhibitors and the substrate D-alanine are changed only slightly, suggesting that the overall substrate binding pocket remains intact. In agreement with data from the pH dependence of ligand binding and with the protein crystal structure, site-directed mutagenesis results emphasize the importance of residue Y238 in controlling access to the active site instead of a role in the substrate/ligand interaction.Keywords: active site lid; function-structure relationships; flavoprotein; reaction mechanism; substrate recognition. D-amino acid oxidase (DAAO; EC 1.4.3.3), an FADcontaining flavoprotein, catalyses dehydrogenation of the D-isomer of amino acids to give the corresponding a-imino acids and, after subsequent hydrolysis, a-keto acids and ammonia. The reduced FAD is then reoxidized by molecular oxygen to yield hydrogen peroxide. The DAAO reaction has many biotechnological applications. Industrially its main use is to remove the side chain of cephalosporin c to give 7-aminocephalosporanic acid, a key intermediate for the production of semisynthetic cephalosporin antibiotics [1]. A fundamental question remains within the large class of flavoprotein oxidases that catalyse the oxidation of amino or a-hydroxy acids regarding the mechanism by which a proton and two electrons are transferred from the substrate a-carbon to the flavin N(5) position during the reductive half-reaction. The precise mechanism of substrate dehydrogenation by DAAO is widely debated, even if the crystal structures of the enzyme purified from pig kidney (pkDAAO) and of the enzyme from Rhodotorula gracilis (RgDAAO) (at a resolution of 2.6 Å and 1.2 Å , respectively) have been determined [2][3][4]. Over the years, three main but different mechanisms have been proposed for the reaction catalysed by this flavoenzyme (reviewed in [5]): (a) a direct hydride-transfer mechanism of a-hydrogen of the substrate to the N(5) position of the flavin [6]; (b) a concerted mechanism in which the a-proton abstraction is coupled with the transfer of a hydride from the amino group of the substrate [7]; and (c) a carbanion mechanism which involves the initial formation of a carbanion by subtracting the a-H of the substrate as a proton [8]. Thus, to deprotonate the a-proton...

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The hair follicle represents a very attractive organ system for studying the precise balance between cell proliferation, growth, differentiation, and death of cells, because it periodically and regularly regenerates, retaining its morphogenetic signals throughout its life. One of the most intriguing oncogenes which is able to induce both cell growth and apoptosis, depending upon the environmental conditions, is c-myc. The aim of the present study was to investigate its presence and localization in human hair follicles by immunohistochemistry and immunofluorescence. Our observations demonstrated the consistent presence of two clusters of cMyc-expressing cells in anagen follicles, located in two annular regions of the inner root sheath, at the border between cells characterized by putative trichohyalin granules and cells which are keratinized. The lower group belongs to Henle's layer, while the upper group belongs to Huxley's layer. cMyc oncoprotein seems to favour apoptosis/differentiation and may be a marker for terminal differentiation of trichocytes, at least in the inner root sheath. Our findings agree with the interpretation that the complex morphology of the hair follicle reflects its complex function; the extrusion of a highly organized multicellular structure, the hair shaft, driven by another highly organized multicellular structure, the inner root sheath.