Flavin-containing Baeyer-Villiger monooxygenases employ NADPH and molecular oxygen to catalyze the insertion of an oxygen atom into a carbon-carbon bond of a carbonylic substrate. These enzymes can potentially be exploited in a variety of biocatalytic applications given the wide use of Baeyer-Villiger reactions in synthetic organic chemistry. The catalytic activity of these enzymes involves the formation of two crucial intermediates: a flavin peroxide generated by the reaction of the reduced flavin with molecular oxygen and the ''Criegee'' intermediate resulting from the attack of the flavin peroxide onto the substrate that is being oxygenated. The crystal structure of phenylacetone monooxygenase, a Baeyer-Villiger monooxygenase from the thermophilic bacterium Thermobifida fusca, exhibits a two-domain architecture resembling that of the disulfide oxidoreductases. The active site is located in a cleft at the domain interface. An arginine residue lays above the flavin ring in a position suited to stabilize the negatively charged flavin-peroxide and Criegee intermediates. This amino acid residue is predicted to exist in two positions; the ''IN'' position found in the crystal structure and an ''OUT'' position that allows NADPH to approach the flavin to reduce the cofactor. Domain rotations are proposed to bring about the conformational changes involved in catalysis. The structural studies highlight the functional complexity of this class of flavoenzymes, which coordinate the binding of three substrates (molecular oxygen, NADPH, and phenylacetone) in proximity of the flavin cofactor with formation of two distinct catalytic intermediates.flavoenzyme ͉ mechanism of catalysis ͉ biocatalysis ͉ Baeyer-Villiger reaction ͉ crystallography A t the end of the 19th century, Baeyer and Villiger (1) discovered that cyclic ketones react with oxidants, such as peroxymonosulfuric acid, to yield lactones. The mechanism of the Baeyer-Villiger reaction involves a nucleophilic attack of a peroxide to the ketone reagent to generate the so-called ''Criegee'' intermediate ( Fig. 1), which is followed by an intramolecular rearrangement that leads to the migration of an alkyl group to an oxygen atom, generating the lactone product. Baeyer-Villiger reactions are of enormous value in synthetic organic chemistry, and the number of their applications is countless.Several microorganisms produce enzymes capable to catalyze Baeyer-Villiger reactions. These proteins are extensively studied for their exploitation in biocatalytic applications (2, 3). This interest follows the problems related to the toxicity and instability of the oxidizing reactants that are currently being used in chemical processes. In addition, enzymatic reactions exhibit a superior degree of enantio-and regioselectivity. Baeyer-Villiger monooxygenases are classified depending on the nature of their flavin cofactor (4). The type I enzymes are the most extensively investigated (5). They are FAD-dependent proteins that use NADPH and molecular oxygen to insert an oxygen atom into th...

Flavin-containing monooxygenases (FMOs) are, after cytochromes P450, the most important monooxygenase system in humans and are involved in xenobiotics metabolism and variability in drug response. The x-ray structure of a soluble prokaryotic FMO from Methylophaga sp. strain SK1 has been solved at 2.6-Å resolution and is now the protein of known structure with the highest sequence similarity to human FMOs. The structure possesses a two-domain architecture, with both FAD and NADP ؉ well defined by the electron density maps. Biochemical analysis shows that the prokaryotic enzyme shares many functional properties with mammalian FMOs, including substrate specificity and the ability to stabilize the hydroperoxyflavin intermediate that is crucial in substrate oxygenation. On the basis of their location in the structure, the nicotinamide ring and the adjacent ribose of NADP ؉ turn out to be an integral part of the catalytic site being actively engaged in the stabilization of the oxygenating intermediate. This feature suggests that NADP(H) has a moonlighting role, in that it adopts two binding modes that allow it to function in both flavin reduction and oxygen reactivity modulation, respectively. We hypothesize that a relative domain rotation is needed to bring NADP(H) to these distinct positions inside the active site. Localization of mutations in human FMO3 that are known to cause trimethylaminuria (fish-odor syndrome) in the elucidated FMO structure provides a structural explanation for their biological effects.drug metabolism ͉ trimethylaminuria ͉ oxygen

Dioxygen (O2) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O2 uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O2 diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O2 diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O 2 from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O 2 reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.computational biochemistry ͉ enzymology ͉ flavin ͉ oxygen reactivity

p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADHdependent FMN reductase and a monooxygenase (C 2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket that binds reduced FMN without large conformational changes. The Phe-266 side chain swings out to provide the space for binding p-hydroxyphenylacetate that is oriented orthogonal to the flavin ring. The geometry of the substrate-binding site of C 2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C 2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C 2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-Å radius and a 29-Å 3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. F lavoprotein monooxygenases use dioxygen to insert an oxygen atom into a substrate and have been found to be involved in a wide variety of biological reactions (1-3). The fundamental property of these enzymes is their ability to promote formation and stabilization of the C4a-hydroperoxyflavin (Fig. 1a) resulting from the reaction of the protein-bound reduced flavin with dioxygen. This key intermediate donates an oxygen atom to the substrate, generating the unstable C4a-hydroxyflavin that eliminates one molecule of water to yield oxidized flavin (5). Understanding the structural bases governing functional properties of monooxygenases is crucial to address one of the most fascinating issues in flavoenzymology: the ability of flavoenzymes to differentially react with molecular oxygen.In recent years, a new group of flavoprotein monooxygenases has been identified. These enzymes consist of two components: a reductase generating reduced flavin and a hydroxylase using reduced flavin to catalyze substrate monooxygenation (6). phydroxyphenylacetate hydroxylase from Acinetobacter baumannii catalyzes hydroxylation of p-hydroxyphenylacetate (HPA) to 3,4-dihydroxyphenylacetate (Fig. 1a). HPA hydroxylase has unusual features in both sequence and catalysis. The smaller reductase component of HPA hydroxylase (C 1 ) performs HPA-stimulated NADH-dependent reduction of free FMN, which is subsequently transferred to the larger monooxygenase component (C 2 ) and used for reaction with dioxygen and HPA monooxygenation (Fig. 1b). Specificity for FMN is conferred by C 1 , whereas C 2 works equally well with both reduced FMN (FMNH Ϫ ; Fig. 1a) and reduced FAD (7-10). C 2 can effectively stabilize the C4a-hydroperoxyflavin intermediate for minutes, and, at high concentration of HPA, a stable dead-end complex between C4a-hydroxyflavin and HPA is observed.Here, we present crystal structures of C 2 in the apoenzyme form and of its complexes with FMNH Ϫ (C 2 :FMNH Ϫ ) and HPA (C 2 :FMNH Ϫ :HPA). Structural analysis reported here provides a fram...

Polarized epithelia form by oriented cell divisions in which the mitotic spindle aligns parallel to the epithelial plane. To orient the mitotic spindle, cortical cues trigger the recruitment of NuMA-dynein-based motors, which pull on astral microtubules via the protein LGN. We demonstrate that the junctional protein Afadin is required for spindle orientation and correct epithelial morphogenesis of Caco-2 cysts. Molecularly, Afadin binds directly and concomitantly to F-actin and to LGN. We determined the crystallographic structure of human Afadin in complex with LGN and show that it resembles the LGN-NuMA complex. In mitosis, Afadin is necessary for cortical accumulation of LGN and NuMA above the spindle poles, in an F-actin-dependent manner. Collectively, our results depict Afadin as a molecular hub governing the enrichment of LGN and NuMA at the cortex. To our knowledge, Afadin is the first-described mechanical anchor between dynein and cortical F-actin.

Coupling of spindle orientation to cellular polarity is a prerequisite for epithelial asymmetric cell divisions. The current view posits that the adaptor Inscuteable (Insc) bridges between Par3 and the spindle tethering machinery assembled on NuMA∶LGN∶Gαi GDP , thus triggering apico-basal spindle orientation. The crystal structure of the Drosophila ortholog of LGN (known as Pins) in complex with Insc reveals a modular interface contributed by evolutionary conserved residues. The structure also identifies a positively charged patch of LGN binding to an invariant EPE-motif present on both Insc and NuMA. In vitro competition assays indicate that Insc competes with NuMA for LGN binding, displaying a higher affinity, and that it is capable of opening the LGN conformational switch. The finding that Insc and NuMA are mutually exclusive interactors of LGN challenges the established model of force generators assembly, which we revise on the basis of the newly discovered biochemical properties of the intervening components.A symmetric cell divisions regulate the position and the fate choice of daughter cells, with impact on numerous phenotypes of multicellular organisms. During development, asymmetric divisions coordinate cell growth with cell specification to determine tissue morphogenesis, while in adult life they sustain tissue homeostasis and regeneration (1). In asymmetric divisions specific cortical landmarks instruct the orientation of the mitotic spindle to promote unequal partitioning of fate determinants in cellular systems as diverse as Caenorhabditis elegans zygotes, Drosophila neuroblasts, as well as vertebrate skin and neural progenitors (2). Spindle coupling to polarity cues involves the recruitment at cortical sites of molecular devices, known as force generators, whose main task is to capture astral microtubules emanating from the spindle poles and to establish pulling forces. Core components of force generators are the evolutionary conserved NuMA∶LGN∶Gαi complexes, termed Mud∶Pins∶Gαi in flies. Topologically, tetratricopeptide repeats (TPR) present in the N-terminal portion of LGN mediate the interactions with NuMA, while GoLoco motifs at the C terminus serve as a docking platform for four Gαi GDP subunits anchored at the plasma membrane via a myristoyl group (3).LGN exclusively binds to GDPloaded Gαi (4). The association of cortical NuMA with the microtubule motor Dynein/Dynactin (5) provides a sliding anchorage for depolymerizing microtubules, whose shrinkage pulls towards the cortex the connected spindle pole. FRET studies revealed that LGN behaves as a conformational switch held in a closed form in interphase by head-to-tail interactions (3).An issue intimately related to how force generators are assembled is how they are recruited at sites of polarization. In polarized asymmetric divisions, the apico-basal polarity axis is established by the asymmetrical distribution of Par3∶Par6∶aPKC at the apical cortex, which are able to recruit NuMA∶LGN∶Gαi GDP via an adaptor named Inscuteable (Insc) (6). Insc w...

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Arabidopsis thalianaglutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+increase inArabidopsisseedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference forl-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology.