In vertebrates, symmetric versus asymmetric cleavage of ␤-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan ␤,␤-carotene-15,15-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving ␤-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of ␤-carotene at the 9,10 double bond of ␤-carotene and resulting in the formation of ␤-apo-10-carotenal and ␤-ionone, a substance known as a floral scent from roses, for example. Besides ␤-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the ␤,␤-carotene-15,15-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency in humans. These plant-derived compounds must be cleaved and metabolically converted by intrinsic carotenoid oxygenases to support the panoply of vitamin A-dependent physiological processes. Two different carotenoid-cleaving enzymes were identified in mammals, the classical carotenoid-15,15-oxygenase (CMO1) and a putative carotenoid-9,10-oxygenase (CMO2). To analyze the role of CMO1 in mammalian physiology, here we disrupted the corresponding gene by targeted homologous recombination in mice. On a diet providing ␤-carotene as major vitamin A precursor, vitamin A levels fell dramatically in several tissues examined. Instead, this mouse mutant accumulated the provitamin in large quantities (e.g. as seen by an orange coloring of adipose tissues). Besides impairments in ␤-carotene metabolism, CMO1 deficiency more generally interfered with lipid homeostasis. Even on a vitamin A-sufficient chow, CMO1 ؊/؊ mice developed a fatty liver and displayed altered serum lipid levels with elevated serum unesterified fatty acids. Additionally, this mouse mutant was more susceptible to high fat diet-induced impairments in fatty acid metabolism. Quantitative reverse transcription-PCR analysis revealed that the expression of peroxisome proliferator-activated receptor ␥-regulated marker genes related to adipogenesis was elevated in visceral adipose tissues. Thus, our study identifies CMO1 as the key enzyme for vitamin A production and provides evidence for a role of carotenoids as more general regulators of lipid metabolism.Dietary lipids are precursors for signaling molecules that control many facets in cell physiology. As the classic example, fat-soluble vitamin A (all-trans-retinol) is essential for processes ranging from development to vision and cell proliferation (1-3). Retinol is the precursor for at least two critical metabolites, 11-cis-retinal, the chromophore of visual G-protein-coupled receptors (4), and retinoic acid (RA).5 Alltrans-RA and 9-cis-RA regulate gene expression via heterodimeric nuclear receptors consisting of an RA receptor and a retinoid X receptor (RXR) (5, 6). Both are ligand-dependent transcription factors belonging to the superfamily of nuclear hormone receptors (7). Additionally, RXRs form heterodimers with other members of the nuclear receptor family (8), including the peroxisome proliferator-activated receptors (PPARs).Because animals, including humans, are unable to synthesize vitamin A de novo, all retinoids (vitamin A and its derivatives) derive from the oxidative cleavage of dietary provitamin A carotenoids, mainly ␤-carotene (9 -11). How this conversion of ␤-carotene occurs (centric and/or eccentric cleavage) is still a matter of debate (12)(13)(14). Recently, two different carotenoidmonooxygenases, CMO1 and CMO2, were molecularly identified in animals, including humans (15). Both belong to a family of structurally related nonheme iron oxygenases, common to all...

The uptake of dietary lipids from the small intestine is a complex process that depends on the activities of specific membrane receptors with yet unknown regulatory mechanisms. Using both mouse models and human cell lines, we show here that intestinal lipid absorption by the scavenger receptor class B type 1 (SR-BI) is subject to control by retinoid signaling. Retinoic acid via retinoic acid receptors induced expression of the intestinal transcription factor ISX. ISX then repressed the expression of SR-B1 and the carotenoid-15,15'-oxygenase Bcmo1. BCMO1 acts downstream of SR-BI and converts absorbed beta,beta-carotene to the retinoic acid precursor, retinaldehyde. Using BCMO1-knockout mice, we demonstrated increased intestinal SR-BI expression and systemic beta,beta-carotene accumulation. SR-BI-dependent accumulation of beta,beta-carotene was prevented by dietary retinoids that induced ISX expression. Thus, our study revealed a diet-responsive regulatory network that controls beta,beta-carotene absorption and vitamin A production by negative feedback regulation. The role of SR-BI in the intestinal absorption of other dietary lipids, including cholesterol, fatty acids, and tocopherols, implicates retinoid signaling in the regulation of lipid absorption more generally and has clinical implications for diseases associated with dyslipidemia.

Background: Mammalian genomes encode two carotenoid oxygenases, but their contributions to vitamin A homeostasis remain undefined. Results: Mammals employ symmetric and eccentric cleaving carotenoid oxygenases to convert different provitamin A carotenoids to vitamin A. Conclusion: Both carotenoid oxygenases contribute to vitamin A production. Significance: Carotenoids are the major source for vitamin A in the human diet.

Evidence from cell culture studies indicates that β-carotene-(BC)-derived apocarotenoid signaling molecules can modulate the activities of nuclear receptors that regulate many aspects of adipocyte physiology. Two BC metabolizing enzymes, the BC-15,15′-oxygenase (Bcmo1) and the BC-9′,10′-oxygenase (Bcdo2) are expressed in adipocytes. Bcmo1 catalyzes the conversion of BC into retinaldehyde and Bcdo2 into β-10′-apocarotenal and β-ionone. Here we analyzed the impact of BC on body adiposity of mice. To genetically dissect the roles of Bcmo1 and Bcdo2 in this process, we used wild-type and Bcmo1 -/- mice for this study. In wild-type mice, BC was converted into retinoids. In contrast, Bcmo1-/- mice showed increased expression of Bcdo2 in adipocytes and β-10′-apocarotenol accumulated as the major BC derivative. In wild-type mice, BC significantly reduced body adiposity (by 28%), leptinemia and adipocyte size. Genome wide microarray analysis of inguinal white adipose tissue revealed a generalized decrease of mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) target genes. Consistently, the expression of this key transcription factor for lipogenesis was significantly reduced both on the mRNA and protein levels. Despite β-10′-apocarotenoid production, this effect of BC was absent in Bcmo1-/- mice, demonstrating that it was dependent on the Bcmo1-mediated production of retinoids. Our study evidences an important role of BC for the control of body adiposity in mice and identifies Bcmo1 as critical molecular player for the regulation of PPARγ activity in adipocytes

The key enzyme responsible for beta-carotene conversion into retinal is beta-carotene 15,15'-monoxygenase (BCMO1). Since it has been reported that the conversion of beta-carotene into vitamin A is highly variable in up to 45% of healthy individuals, we hypothesized that genetic polymorphisms in the BCMO1 gene could contribute to the occurrence of the poor converter phenotype. Here we describe the screening of the total open reading frame of the BCMO1 coding region that led to the identification of two common nonsynonymous single nucleotide polymorphisms (R267S: rs12934922; A379V: rs7501331) with variant allele frequencies of 42 and 24%, respectively. In vitro biochemical characterization of the recombinant 267S + 379V double mutant revealed a reduced catalytic activity of BCMO1 by 57% (P<0.001). Assessment of the responsiveness to a pharmacological dose of beta-carotene in female volunteers confirmed that carriers of both the 379V and 267S + 379V variant alleles had a reduced ability to convert beta-carotene, as indicated through reduced retinyl palmitate:beta-carotene ratios in the triglyceride-rich lipoprotein fraction [-32% (P=0.005) and -69% (P=0.001), respectively] and increased fasting beta-carotene concentrations [+160% (P=0.025) and +240% (P=0.041), respectively]. Our data show that there is genetic variability in beta-carotene metabolism and may provide an explanation for the molecular basis of the poor converter phenotype within the population.

The egg yolk of vertebrates contains carotenoids, which account for its characteristic yellow color in some species. Such plant-derived compounds, e.g. β-carotene, serve as the natural precursors (provitamins) of vitamin A, which is indispensable for chordate development. As egg yolk also contains stored vitamin A, carotenoids have so far been solely discussed as pigments for the coloration of the offspring. Based on our recent molecular identification of the enzyme catalyzing provitamin A conversion to vitamin A, we address a possible role of provitamin A during zebrafish (Danio rerio) development. We cloned the zebrafish gene encoding the vitamin A-forming enzyme, a β,β-carotene-15,15′-oxygenase. Analysis of its mRNA expression revealed that it is under complex spatial and temporal control during development. Targeted

There is an urgent need to develop new antimicrobial agents due to increasing bacterial resistance to therapeutically used drugs. Most methicillin-resistent Staphylococcus aureus (MRSA) strains are resistent not only to b-lactams, but also to most other antimicrobial agents.1) Penicillin resistance among Streptococcus pneumoniae strains is widely accepted as a global problem. [2][3][4][5] Bacteria have developed several strategies for escaping the lethal action of b-lactams. It may be expected that specific circumstance will make one the more effective stragegy than the other. 6) Much effort has been devoted to the discovery of drugs which would not be cleaved by b-lactamases of pathogenic strains and which have suitable physicochemical and pharmacodynamic profiles. 7,8) The modifications of b-lactam antibiotics could not keep pace with the development of resistance in the pathogenic microorganisms, so that numerous bacteria, among them multidrug resistant Staphylococcus strains, can no longer be treated with the currently available b-lactam antibiotics. 1,9,10) Besides the modification of existing antibiotics by chemical or biochemical methods the coupling of presently used antibiotics with other bioactive compounds or components from them which are not in use till now is a promising way to generate novel molecules with improved therapeutic properties.Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2), classically considered a hydroquinone oxidizing enzyme, is able to oligomerize molecules. Up to now main application fields of this enzyme are waste detoxification, textile dye transformation, biosensors and diagnostic application, where the capability to catalyze polymerization reactions is used. 11-13)Recently we reported about our synthetic results on coupling reactions with laccase.14-17) Now we have employed laccase to achieve derivatisation of b-lactam antibiotics and to couple them with derivatives of 2,5-dihydroxybenzoic acid. These derivatives are structurally related to the ganomycins, a new chemical class of antibacterial compounds 18) and to other antibacterial active isolates 19,20) therefore interesting as coupling partner for b-lactams using laccase as initiator of the reaction to produce novel hybridantibiotics by biotransformation.The use of laccase for the derivatisation of antibiotics is limited to a few examples including the phenolic oxidation of 7-(4-hydroxyphenylacetamido)cephalosporinic acid, 21) the dimerization of penicillin X 22) and the oxidative coupling of hydroquinone and mithramicine. 23) In the examples realized to date, the sought object of enhancement of the bioactive effect has not been achieved. 21-23)The aim of this study was (i) to investigate whether laccase can be used for the synthesis of novel penicillins by heteromolecular coupling of two different compounds, (ii) to characterize the products of the reaction, and (iii) to analyze the biological activity of the novel penicillins. Results and DiscussionBiotransformation of Amoxicillin and Ampicillin by Laccase of Tr...