D-Amino acid oxidase (DAAO) is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-isomers of neutral and polar amino acids. This enzymatic activity has been identified in most eukaryotic organisms, the only exception being plants. In the various organisms in which it does occur, DAAO fulfills distinct physiological functions: from a catabolic role in yeast cells, which allows them to grow on D-amino acids as carbon and energy sources, to a regulatory role in the human brain, where it controls the levels of the neuromodulator D-serine. Since 1935, DAAO has been the object of an astonishing number of investigations and has become a model for the dehydrogenase-oxidase class of flavoproteins. Structural and functional studies have suggested that specific physiological functions are implemented through the use of different structural elements that control access to the active site and substrate/product exchange. Current research is attempting to delineate the regulation of DAAO functions in the contest of complex biochemical and physiological networks.

Chloroplast ferredoxin-NADP؉ reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP ؉ photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2-phosphoryl group of NADP ؉ , replacement of the C-terminal tyrosine (Tyr 308 in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k cat value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD ؉ complexes revealed that the nicotinamide ring of NAD ؉ binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k cat values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP ؉ reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.

To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 microM-1 s-1) due to a substantial decrease of kcat. NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (Kd = 37 microM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD-NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.

Ferredoxin-NADP؉ reductase, the prototype of a large family of structurally related flavoenzymes, pairs single electrons carried by ferredoxin I and transfers them as a hydride to NADP ؉ . Four mutants of the enzyme, in which Glu-312 was replaced with Asp, Gln, Leu, and Ala to probe the role of the residue charge, size, and polarity in the enzyme activity, have been heterologously expressed, purified, and characterized through steadystate, rapid kinetic studies, ligand-binding experiments, and three-dimensional structure determination by x-ray crystallography. The E312L mutant was the only one that was almost inactive (ϳ1%), whereas unexpectedly the E312A reductase was 10 -100% active with the various acceptors tested. Rapid kinetic absorption spectroscopy studies demonstrated that flavin reduction by NADPH was impaired in the mutants. Furthermore, NADP(H) binding was partially perturbed. These functional and structural studies lead us to conclude that Glu-312 does not fulfil the role of proton donor during catalysis, but it is required for proper binding of the nicotinamide ring of NADP(H). In addition, its charge modulates the two one-electron redox potentials of the flavin to stabilize the semiquinone form. Ferredoxin-NADPϩ reductase (FNR) 1 from plants and cyanobacteria fulfils the role of electrical switch between one-and two-electron transfer processes during NADP ϩ photoreduction in the photosynthetic electron transport chain (1, 2). FNR became the structural prototype of a large family of structurally related flavoenzymes since the resolution of its three-dimensional structure, which highlighted a novel flavin binding fold (3). Indeed, FNR-like modules are building blocks for constructing both simple and complex flavoproteins with the most varied biological functions not only in prokaryotes and plants but also in animals (4, 5). Generally, the members of the family are highly specific either for NAD ϩ or NADP ϩ , whereas they are more permissive with respect to the electron acceptor.The non-physiological reactions catalyzed in vitro by FNR can be divided into two half-reactions corresponding to transfer of a hydride between NADPH and FAD, and transfer of single electrons between reduced FAD and electron carriers (A), such as Fd (cytochrome c), ferricyanide, and presumably INT, according to Scheme 1.The stoichiometric coefficient n can be 1 or 2, depending on the type of electron acceptor. The reductive half-reaction of FNR and other FNR family members occurs in discrete steps, which involve two Michaelis complexes (MC) and two chargetransfer complexes (CT) (6 -10), according to Scheme 2.In order to clarify the mechanism of action of hydride transfer mediated by the enzyme through flavin-nicotinamide rings interaction, for which there was no support from crystal structures (3, 11, 12), our group and others have carried out sitedirected mutagenesis of the five conserved residues surrounding the isoalloxazine ring in the active center of FNR: Tyr-95, Ser-96, Cys-272, Glu-312, and Tyr-314 (spinach numbering)...

Cholesterol oxidase is a bacterial FAD‐containing flavooxidase that catalyzes the first reaction in cholesterol catabolism. Indeed, this enzyme catalyzes two reactions: the oxidation of the C3‐OH group of cholesterol (and other sterols) to give cholest‐5‐en‐3‐one; and its isomerization to cholest‐4‐en‐3‐one. In the past several years, the structural and functional characterization of cholesterol oxidase has been developed together with its application as a biological tool. Cholesterol oxidase has been used in biocatalysis for the production of a number of steroids, as an insecticidal protein against boll weevil larvae and, in particular, as a diagnostic enzyme for determining serum levels of cholesterol. These applications prompted various laboratories worldwide to isolate this flavooxidase from different sources and to improve its properties by protein engineering, further increasing our knowledge on its structure–function relationships. These studies also discovered new physiological roles for cholesterol oxidase (e.g. in virulence and as an antifungal sensor). We assume that the investigations of cholesterol oxidase and its applications will continue to grow quickly in the near future, in particular to uncover unexpected, new areas of application.

Basic leucine zipper (bZIP) transcription factors are dimers that recognize mainly palindromic DNA sites. It has been assumed that bZIP factors have to form a dimer in order to bind to their target DNA. We find that DNA binding of both monomeric and dimeric bZIP transcription factor GCN4 is diffusion-limited and that, therefore, the rate of dimerization of the bZIP domain does not affect the rate of DNA recognition and GCN4 need not dimerize in order to bind to its specific DNA site. The results have implications for the mechanism by which bZIP transcription factors find their target sites for transcriptional regulation.z 1998 Federation of European Biochemical Societies.

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a ⌬loop mutant, in which 14 residues belonging to a loop connecting strands ␤F5-␤F6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH 4 SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the ␤F5-␤F6 loop of one monomer and the ␣I3Ј and ␣I3Љ helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric ⌬loop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T m s) of 48°C and 54°C, respectively). The absence of complications ensuing from association equilibria in the mutant ⌬loop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release. Keywords: Flavoprotein; lipophilic ions; folding; dimerization; energetic domains; structural domains; quaternary structure; thermal stability Supplemental material: See www.proteinscience.org.Flavoproteins are a group of enzymes that catalyze a variety of reactions and that are characterized by the presence of a flavin cofactor. Concerning the coenzyme binding, flavoproteins divide into three classes: those possessing the coenzyme covalently linked through the isoalloxazine ring to a histidine, cysteine, or tyrosine residue of the polypeptide chain (Decker 1991); those in which the flavin is noncovalently bound; and those that use flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a substrate. In the first two flavoprotein classes, attainment of the catalytic activity requires binding of the cofactor to the apoprotein, which generally has a different conformation from the holoenzyme (van Mierlo and Steensma 2000).Cofactor binding is of paramount importance in vivo, because many flavoenzymes are nuclear encoded, synthesized as larger precursors on free cytosolic ribosomes, and Reprint requests to: Francesco Bonomi, DISMA, University of Milan, via Celoria 2, 20133 Milan, Italy; e-mail: francesco.bonomi@unimi.it; fax: 39-02-50316801.Abbreviations: DAAO...

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