Cleavage of resveratrol in fungi: characterization of the enzyme Rco1 from Ustilago maydis.

Ustilago maydis, the causative agent of corn smut disease, contains two genes encoding members of the carotenoid cleavage oxygenase family, a group of enzymes that cleave double bonds in different substrates. One of them, Cco1, was formerly identified as a ß-carotene cleaving enzyme. Here we elucidate the function of the protein encoded by the second gene, termed here as Ustilago maydis Resveratrol cleavage oxygenase 1 (Um Rco1). In vitro incubations of heterologously expressed and purified UM Rco1 with different carotenoid and stilbene substrates demonstrate that it cleaves the interphenyl Cα-Cß double bond of the phytoalexin resveratrol and its derivative piceatannol. Um Rco1 exhibits a high degree of substrate specificity, as suggested by the lack of activity on carotenoids and the other resveratrol-related compounds tested. The activity of Um Rco1 was confirmed by incubation of U. maydis rco1 deletion and over-expression strains with resveratrol. Furthermore, treatment with resveratrol resulted in striking alterations of cell morphology. However, pathogenicity assays indicated that Um rco1 is largely dispensable for biotrophic development. Our work reveals Um Rco1 as the first eukaryotic resveratrol cleavage enzyme identified so far. Moreover, Um Rco1 represents a subfamily of fungal enzymes likely involved in the degradation of stilbene compounds, as suggested by the cleavage of resveratrol by homologs from Aspergillus fumigatus, Chaetomium globosum and Botryotinia fuckeliana.

The Mycobacterium tuberculosis ORF Rv0654 encodes a carotenoid oxygenase mediating central and excentric cleavage of conventional and aromatic carotenoids.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is assumed to lack carotenoids, which are widespread pigments fulfilling important functions as radical scavengers and as a source of apocarotenoids. In mammals, the synthesis of apocarotenoids, including retinoic acid, is initiated by the ß-carotene cleavage oxygenases I and II catalyzing either a central or an excentric cleavage of ß-carotene, respectively. The M. tuberculosis ORF Rv0654 codes for a putative carotenoid oxygenase conserved in other mycobacteria. In the present study, we investigated the corresponding enzyme, here named M. tuberculosis carotenoid cleavage oxygenase (MtCCO). Using heterologously expressed and purified protein, we show that MtCCO converts several carotenoids and apocarotenoids in vitro. Moreover, the identification of the products suggests that, in contrast to other carotenoid oxygenases, MtCCO cleaves the central C15-C15' and an excentric double bond at the C13-C14 position, leading to retinal (C(20)), ß-apo-14'-carotenal (C(22)) and ß-apo-13-carotenone (C(18)) from ß-carotene, as well as the corresponding hydroxylated products from zeaxanthin and lutein. Moreover, the enzyme cleaves also 3,3'-dihydroxy-isorenieratene representing aromatic carotenoids synthesized by other mycobacteria. Quantification of the products from different substrates indicates that the preference for each of the cleavage positions is determined by the hydroxylation and the nature of the ionone ring. The data obtained in the present study reveal MtCCO to be a novel carotenoid oxygenase and indicate that M. tuberculosis may utilize carotenoids from host cells and interfere with their retinoid metabolism.

Novel apocarotenoid intermediates in Neurospora crassa mutants imply a new biosynthetic reaction sequence leading to neurosporaxanthin formation.

Neurosporaxanthin, beta-apo-4'-carotenoic acid (C35), represents the end-product of the carotenoid pathway in Neurospora crassa. It is supposed to be synthesized in three steps catalyzed by sequential AL-2, CAO-2 and YLO-1 activities: (i) cyclization of 3,4-didehydrolycopene (C40); (ii) cleavage of torulene into beta-apo-4'-carotenal (C35); and finally (iii) oxidation of beta-apo-4'-carotenal. However, analyses of the ylo-1 mutant revealed the accumulation of intermediates other than beta-apo-4'-carotenal. Here, we generated a 3,4-didehydrolycopene accumulating Escherichia coli strain and showed that CAO-2 cleaves this acyclic carotene in vivo and in vitro yielding apo-4'-lycopenal. The apocarotenoids accumulated in the ylo-1 mutant were then identified as apo-4'-lycopenal and apo-4'-lycopenol, pointing to the former as the YLO-1 substrate and indicating that cyclization is the last step in neurosporaxanthin biosynthesis. This was further substantiated by analyses of a cyclase-deficient al-2 mutant, revealing the accumulation of apo-4'-lycopenoic acid. The three acyclic apocarotenoids presented here have not been found naturally before.

The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last reaction in the Neurospora carotenoid pathway.

The accumulation of the apocarotenoid neurosporaxanthin and its carotene precursors explains the orange pigmentation of the Neurospora surface cultures. Neurosporaxanthin biosynthesis requires the activity of the albino gene products (AL-1, AL-2 and AL-3), which yield the precursor torulene. Recently, we identified the carotenoid oxygenase CAO-2, which cleaves torulene to produce the aldehyde beta-apo-4'-carotenal. This revealed a last missing step in Neurospora carotenogenesis, namely the oxidation of the CAO-2 product to the corresponding acid neurosporaxanthin. The mutant ylo-1, which exhibits a yellow colour, lacks neurosporaxanthin and accumulates several carotenes, but its biochemical basis is unknown. Based on available genetic data, we identified ylo-1 in the Neurospora genome, which encodes an enzyme representing a novel subfamily of aldehyde dehydrogenases, and demonstrated that it is responsible for the yellow phenotype, by sequencing and complementation of mutant alleles. In contrast to the precedent structural genes in the carotenoid pathway, light does not induce the synthesis of ylo-1 mRNA. In vitro incubation of purified YLO-1 protein with beta-apo-4'-carotenal produced neurosporaxanthin through the oxidation of the terminal aldehyde into a carboxyl group. We conclude that YLO-1 completes the set of enzymes needed for the synthesis of this major Neurospora pigment.

In vitro characterization of a carotenoid cleavage dioxygenase from Nostoc sp. PCC 7120 reveals a novel cleavage pattern, cytosolic localization and induction by highlight.

Carotenoid oxygenases catalyse the cleavage of C-C double bonds forming apocarotenoids, a diverse group of compounds, including retinoids and the precursors of some phytohormones. Some apocarotenoids, like beta-ionone (C(13)), are ecologically important volatiles released by plants and cyanobacteria. In this work, we elucidated the activity of the Nostoccarotenoid cleavage dioxygenase (NosCCD, previously named NSC1) using synthetic and cyanobacterial substrates. NosCCD converted bicyclic and monocyclic xanthophylls, including myxoxanthophylls, glycosylated carotenoids that are essential for thylakoid and cell wall structure. The products identified revealed two different cleavage patterns. The first is observed with bicyclic xanthophylls and is identical with that of plant orthologues, while the second is novel and occurs upon cleavage of monocyclic substrates at the C9-C10 and C7'-C8' double bonds. These properties enable the enzyme to produce a plenitude of different C(10) and C(13) apocarotenoids. Expression analyses indicated a role of NosCCD in response to highlight stress. Western blot analyses of Nostoc cells revealed NosCCD as a soluble enzyme in the cytosol, which also accomodates NosCCD substrates. Incubation of the corresponding fraction with synthetic substrates revealed the activity of the native enzyme and confirmed its induction by highlight.

Retinal biosynthesis in fungi: characterization of the carotenoid oxygenase CarX from Fusarium fujikuroi.

The car gene cluster of the ascomycete Fusarium fujikuroi encodes two enzymes responsible for torulene biosynthesis (CarRA and CarB), an opsin-like protein (CarO), and a putative carotenoid cleaving enzyme (CarX). It was presumed that CarX catalyzes the formation of the major carotenoid in F. fujikuroi, neurosporaxanthin, a cleavage product of torulene. However, targeted deletion of carX did not impede neurosporaxanthin biosynthesis. On the contrary, DeltacarX mutants showed a significant increase in the total carotenoid content, indicating an involvement of CarX in the regulation of the pathway. In this work, we investigated the enzymatic activity of CarX. The expression of the enzyme in beta-carotene-accumulating Escherichia coli cells led to the formation of the opsin chromophore retinal. The identity of the product was proven by high-performance liquid chromatography and gas chromatography-mass spectrometry. Subsequent in vitro assays with heterologously expressed and purified CarX confirmed its beta-carotene-cleaving activity and revealed its capability to produce retinal also from other substrates, such as gamma-carotene, torulene, and beta-apo-8'-carotenal. Our data indicate that the occurrence of at least one beta-ionone ring in the substrate is required for the cleavage reaction and that the cleavage site is determined by the distance to the beta-ionone ring. CarX represents the first retinal-synthesizing enzyme reported in the fungal kingdom so far. It seems likely that the formed retinal is involved in the regulation of the carotenoid biosynthetic pathway via a negative feedback mechanism.

Retinal is formed from apo-carotenoids in Nostoc sp. PCC7120: in vitro characterization of an apo-carotenoid oxygenase.

The sensory rhodopsin from Anabaena (Nostoc) sp. PCC7120 is the first cyanobacterial retinylidene protein identified. Here, we report on NosACO (Nostoc apo-carotenoid oxygenase), encoded by the ORF (open reading frame) all4284, as the candidate responsible for the formation of the required chromophore, retinal. In contrast with the enzymes from animals, NosACO converts beta-apo-carotenals instead of beta-carotene into retinal in vitro. The identity of the enzymatic products was proven by HPLC and gas chromatography-MS. NosACO exhibits a wide substrate specificity with respect to chain lengths and functional end-groups, converting beta-apo-carotenals, (3R)-3-hydroxy-beta-apo-carotenals and the corresponding alcohols into retinal and (3R)-3-hydroxyretinal respectively. However, kinetic analyses revealed very divergent Km and Vmax values. On the basis of the crystal structure of SynACO (Synechocystis sp. PCC6803 apo-carotenoid oxygenase), a related enzyme showing similar enzymatic activity, we designed a homology model of the native NosACO. The deduced structure explains the absence of beta-carotene-cleavage activity and indicates that NosACO is a monotopic membrane protein. Accordingly, NosACO could be readily reconstituted into liposomes. To localize SynACO in vivo, a Synechocystis knock-out strain was generated expressing SynACO as the sole carotenoid oxygenase. Western-blot analyses showed that the main portion of SynACO occurred in a membrane-bound form.