Bioavailability and provitamin A activity of neurosporaxanthin in mice.

Various species of ascomycete fungi synthesize the carboxylic carotenoid neurosporaxanthin. The unique chemical structure of this xanthophyll reveals that: (1) Its carboxylic end and shorter length increase the polarity of neurosporaxanthin in comparison to other carotenoids, and (2) it contains an unsubstituted ß-ionone ring, conferring the potential to form vitamin A. Previously, neurosporaxanthin production was optimized in Fusarium fujikuroi, which allowed us to characterize its antioxidant properties in in vitro assays. In this study, we assessed the bioavailability of neurosporaxanthin compared to other provitamin A carotenoids in mice and examined whether it can be cleaved by the two carotenoid-cleaving enzymes: ß-carotene-oxygenase 1 (BCO1) and 2 (BCO2). Using Bco1-/-Bco2-/- mice, we report that neurosporaxanthin displays greater bioavailability than ß-carotene and ß-cryptoxanthin, as evidenced by higher accumulation and decreased fecal elimination. Enzymatic assays with purified BCO1 and BCO2, together with feeding studies in wild-type, Bco1-/-, Bco2-/-, and Bco1-/-Bco2-/- mice, revealed that neurosporaxanthin is a substrate for either carotenoid-cleaving enzyme. Wild-type mice fed neurosporaxanthin displayed comparable amounts of vitamin A to those fed ß-carotene. Together, our study unveils neurosporaxanthin as a highly bioavailable fungal carotenoid with provitamin A activity, highlighting its potential as a novel food additive.

ASTER-B regulates mitochondrial carotenoid transport and homeostasis.

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by ß-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon ß-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by ß-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-ß-cyclodextrin treatment enhanced zeaxanthin but not ß-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.

Mode of autophosphorylation in bacteriophytochromes RpBphP2 and RpBphP3.

Phytochromes are red-light photoreceptors that regulate a wide range of physiological processes in plants, fungi and bacteria. Canonical bacteriophytochromes are photosensory histidine kinases that undergo light-dependent autophosphorylation, thereby regulating cellular responses to red light via two-component signaling pathways. However, the molecular mechanism of kinase activation remains elusive for bacteriophytochromes. In particular, the directionality of autophosphorylation is still an open question in these dimeric photoreceptor kinases. In this work, we perform histidine kinase assays on two tandem bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris. By examining the kinase activities of full-length bacteriophytochromes and two loss-of-function mutants under different light conditions, we demonstrate that RpBphP2 and RpBphP3 undergo light-dependent trans-phosphorylation between protomers in both homodimeric and heterodimeric forms. We have further determined the crystal structure of the histidine kinase domains of RpBphP2 at 3.19 Å resolution. Based on structural comparisons and homology modeling, we also present a model to account for the actions of trans-autophosphorylation in bacteriophytochromes.

Aster la vista: Unraveling the biochemical basis of carotenoid homeostasis in the human retina.

Carotenoids play pivotal roles in vision as light filters and precursor of chromophore. Many vertebrates also display the colorful pigments as ornaments in bare skin parts and feathers. Proteins involved in the transport and metabolism of these lipids have been identified including class B scavenger receptors and carotenoid cleavage dioxygenases. Recent research implicates members of the Aster protein family, also known as GRAM domain-containing (GRAMD), in carotenoid metabolism. These multi-domain proteins facilitate the intracellular movement of carotenoids from their site of cellular uptake by scavenger receptors to the site of their metabolic processing by carotenoid cleavage dioxygenases. We provide a model how the coordinated interplay of these proteins and their differential expression establishes carotenoid distribution patterns and function in tissues, with particular emphasis on the human retina.

Carotenoid modifying enzymes in metazoans.

Carotenoids constitute an essential dietary component of animals and other non-carotenogenic species which use these pigments in both their modified and unmodified forms. Animals utilize uncleaved carotenoids to mitigate light damage and oxidative stress and to signal fitness and health. Carotenoids also serve as precursors of apocarotenoids including retinol, and its retinoid metabolites, which carry out essential functions in animals by forming the visual chromophore 11-cis-retinaldehyde. Retinoids, such as all-trans-retinoic acid, can also act as ligands of nuclear hormone receptors. The fact that enzymes and biochemical pathways responsible for the metabolism of carotenoids in animals bear resemblance to the ones in plants and other carotenogenic species suggests an evolutionary relationship. We will explore some of the modes of transmission of carotenoid genes from carotenogenic species to metazoans. This apparent relationship has been successfully exploited in the past to identify and characterize new carotenoid and retinoid modifying enzymes. We will review approaches used to identify putative animal carotenoid enzymes, and we will describe methods used to functionally validate and analyze the biochemistry of carotenoid modifying enzymes encoded by animals.

Aster proteins mediate carotenoid transport in mammalian cells.

Some mammalian tissues uniquely concentrate carotenoids, but the underlying biochemical mechanism for this accumulation has not been fully elucidated. For instance, the central retina of the primate eyes displays high levels of the carotenoids, lutein, and zeaxanthin, whereas the pigments are largely absent in rodent retinas. We previously identified the scavenger receptor class B type 1 and the enzyme ß-carotene-oxygenase-2 (BCO2) as key components that determine carotenoid concentration in tissues. We now provide evidence that Aster (GRAM-domain-containing) proteins, recently recognized for their role in nonvesicular cholesterol transport, engage in carotenoid metabolism. Our analyses revealed that the StART-like lipid binding domain of Aster proteins can accommodate the bulky pigments and bind them with high affinity. We further showed that carotenoids and cholesterol compete for the same binding site. We established a bacterial test system to demonstrate that the StART-like domains of mouse and human Aster proteins can extract carotenoids from biological membranes. Mice deficient for the carotenoid catabolizing enzyme BCO2 concentrated carotenoids in Aster-B protein-expressing tissues such as the adrenal glands. Remarkably, Aster-B was expressed in the human but not in the mouse retina. Within the retina, Aster-B and BCO2 showed opposite expression patterns in central versus peripheral parts. Together, our study unravels the biochemical basis for intracellular carotenoid transport and implicates Aster-B in the pathway for macula pigment concentration in the human retina.

Genomic consequences of domestication of the Siamese fighting fish.

Siamese fighting (betta) fish are among the most popular and morphologically diverse pet fish, but the genetic bases of their domestication and phenotypic diversification are largely unknown. We assembled de novo the genome of a wild Betta splendens and whole-genome sequenced 98 individuals across five closely related species. We find evidence of bidirectional hybridization between domesticated ornamental betta and other wild Betta species. We discover dmrt1 as the main sex determination gene in ornamental betta and that it has lower penetrance in wild B. splendens. Furthermore, we find genes with signatures of recent, strong selection that have large effects on color in specific parts of the body or on the shape of individual fins and that most are unlinked. Our results demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication and launch betta as a powerful new system for evolutionary genetics.

Crystal structure of a far-red-sensing cyanobacteriochrome reveals an atypical bilin conformation and spectral tuning mechanism.

Cyanobacteriochromes (CBCRs) are small, linear tetrapyrrole (bilin)-binding photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. More spectrally diverse than canonical red/far-red-sensing phytochromes, CBCRs were thought to be restricted to sensing visible and near UV light until recently when several subfamilies with far-red-sensing representatives (frCBCRs) were discovered. Two of these frCBCRs subfamilies have been shown to incorporate bilin precursors with larger pi-conjugated chromophores, while the third frCBCR subfamily uses the same phycocyanobilin precursor found in the bulk of the known CBCRs. To elucidate the molecular basis of far-red light perception by this third frCBCR subfamily, we determined the crystal structure of the far-red-absorbing dark state of one such frCBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 which exhibits a reversible far-red/orange photocycle. Determined by room temperature serial crystallography and cryocrystallography, the refined 2.7-Å structure reveals an unusual all-Z,syn configuration of the phycocyanobilin (PCB) chromophore that is considerably less extended than those of previously characterized red-light sensors in the phytochrome superfamily. Based on structural and spectroscopic comparisons with other bilin-binding proteins together with site-directed mutagenesis data, our studies reveal protein-chromophore interactions that are critical for the atypical bathochromic shift. Based on these analyses, we propose that far-red absorption in Anacy_2551g3 is the result of the additive effect of two distinct red-shift mechanisms involving cationic bilin lactim tautomers stabilized by a constrained all-Z,syn conformation and specific interactions with a highly conserved anionic residue.

The Structural and Biochemical Basis of Apocarotenoid Processing by ß-Carotene Oxygenase-2.

In mammals, carotenoids are converted by two carotenoid cleavage oxygenases into apocarotenoids, including vitamin A. Although knowledge about ß-carotene oxygenase-1 (BCO1) and vitamin A metabolism has tremendously increased, the function of ß-carotene oxygenase-2 (BCO2) remains less well-defined. We here studied the role of BCO2 in the metabolism of long chain ß-apocarotenoids, which recently emerged as putative regulatory molecules in mammalian biology. We showed that recombinant murine BCO2 converted the alcohol, aldehyde, and carboxylic acid of a ß-apocarotenoid substrate by oxidative cleavage at position C9,C10 into a ß-ionone and a diapocarotenoid product. Chain length variation (C20 to C40) and ionone ring site modifications of the apocarotenoid substrate did not impede catalytic activity or alter the regioselectivity of the double bond cleavage by BCO2. Isotope labeling experiments revealed that the double bond cleavage of an apocarotenoid followed a dioxygenase reaction mechanism. Structural modeling and site directed mutagenesis identified amino acid residues in the substrate tunnel of BCO2 that are critical for apocarotenoid binding and catalytic processing. Mice deficient for BCO2 accumulated apocarotenoids in their livers, indicating that the enzyme engages in apocarotenoid metabolism. Together, our study provides novel structural and functional insights into BCO2 catalysis and establishes the enzyme as a key component of apocarotenoid homeostasis in mice.

An automated platform for in situ serial crystallography at room temperature.

Direct observation of functional motions in protein structures is highly desirable for understanding how these nanomachineries of life operate at the molecular level. Because cryogenic temperatures are non-physiological and may prohibit or even alter protein structural dynamics, it is necessary to develop robust X-ray diffraction methods that enable routine data collection at room temperature. We recently reported a crystal-on-crystal device to facilitate in situ diffraction of protein crystals at room temperature devoid of any sample manipulation. Here an automated serial crystallography platform based on this crystal-on-crystal technology is presented. A hardware and software prototype has been implemented, and protocols have been established that allow users to image, recognize and rank hundreds to thousands of protein crystals grown on a chip in optical scanning mode prior to serial introduction of these crystals to an X-ray beam in a programmable and high-throughput manner. This platform has been tested extensively using fragile protein crystals. We demonstrate that with affordable sample consumption, this in situ serial crystallography technology could give rise to room-temperature protein structures of higher resolution and superior map quality for those protein crystals that encounter difficulties during freezing. This serial data collection platform is compatible with both monochromatic oscillation and Laue methods for X-ray diffraction and presents a widely applicable approach for static and dynamic crystallographic studies at room temperature.

The human mitochondrial enzyme BCO2 exhibits catalytic activity toward carotenoids and apocarotenoids.

The enzyme ß-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzyme's catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9',C10'. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues.

Structural basis of molecular logic OR in a dual-sensor histidine kinase.

Signal detection and integration by sensory proteins constitute the critical molecular events as living organisms respond to changes in a complex environment. Many sensory proteins adopt a modular architecture that integrates the perception of distinct chemical or physical signals and the generation of a biological response in the same protein molecule. Currently, how signal perception and integration are achieved in such a modular, often dimeric, framework remains elusive. Here, we report a dynamic crystallography study on the tandem sensor domains of a dual-sensor histidine kinase PPHK (phosphorylation-responsive photosensitive histidine kinase) that operates a molecular logic OR, by which the output kinase activity is modulated by a phosphorylation signal and a light signal. A joint analysis of ∼170 crystallographic datasets probing different signaling states shows remarkable dimer asymmetry as PPHK responds to the input signals and transitions from one state to the other. Supported by mutational data and structural analysis, these direct observations reveal the working mechanics of the molecular logic OR in PPHK, where the light-induced bending of a long signaling helix at the dimer interface is counteracted by the ligand-induced structural changes from a different sensor domain. We propose that the logic OR of PPHK, together with an upstream photoreceptor, implements a "long-pass" red light response distinct from those accomplished by classical phytochromes.

Crystal-on-crystal chips for in situ serial diffraction at room temperature.

Recent developments in serial crystallography at X-ray free electron lasers (XFELs) and synchrotrons have been driven by two scientific goals in structural biology - first, static structure determination from nano or microcrystals of membrane proteins and large complexes that are difficult for conventional cryocrystallography, and second, direct observations of transient structural species in biochemical reactions at near atomic resolution. Since room-temperature diffraction experiments naturally demand a large quantity of purified protein, sample economy is critically important for all steps of serial crystallography from crystallization, crystal delivery to data collection. Here we report the development and applications of "crystal-on-crystal" devices to facilitate large-scale in situ serial diffraction experiments on protein crystals of all sizes - large, small, or microscopic. We show that the monocrystalline quartz as a substrate material prevents vapor loss during crystallization and significantly reduces background X-ray scattering. These devices can be readily adopted at XFEL and synchrotron beamlines, which enable efficient delivery of hundreds to millions of crystals to the X-ray beam, with an overall protein consumption per dataset comparable to that of cryocrystallography.

Photoactivation mechanism of a carotenoid-based photoreceptor.

Photoprotection is essential for efficient photosynthesis. Cyanobacteria have evolved a unique photoprotective mechanism mediated by a water-soluble carotenoid-based photoreceptor known as orange carotenoid protein (OCP). OCP undergoes large conformational changes in response to intense blue light, and the photoactivated OCP facilitates dissipation of excess energy via direct interaction with allophycocyanins at the phycobilisome core. However, the structural events leading up to the OCP photoactivation remain elusive at the molecular level. Here we present direct observations of light-induced structural changes in OCP captured by dynamic crystallography. Difference electron densities between the dark and illuminated states reveal widespread and concerted atomic motions that lead to altered protein-pigment interactions, displacement of secondary structures, and domain separation. Based on these crystallographic observations together with site-directed mutagenesis, we propose a molecular mechanism for OCP light perception, in which the photochemical property of a conjugated carbonyl group is exploited. We hypothesize that the OCP photoactivation starts with keto-enol tautomerization of the essential 4-keto group in the carotenoid, which disrupts the strong hydrogen bonds between the bent chromophore and the protein moiety. Subsequent structural changes trapped in the crystal lattice offer a high-resolution glimpse of the initial molecular events as OCP begins to transition from the orange-absorbing state to the active red-absorbing state.