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Neurosporaxanthin (β-apo-4′-carotenoic acid) biosynthesis has been studied in detail in the fungus Fusarium fujikuroi. The genes and enzymes for this biosynthetic pathway are known until the last enzymatic step, the oxidation of the aldehyde group of its precursor, β-apo-4′-carotenal. On the basis of sequence homology to Neurospora crassa YLO-1, which mediates the formation of apo-4′-lycopenoic acid from the corresponding aldehyde substrate, we cloned the carD gene of F. fujikuroi and investigated the activity of the encoded enzyme. In vitro assays performed with heterologously expressed protein showed the formation of neurosporaxanthin and other apocarotenoid acids from the corresponding apocarotenals. To confirm this function in vivo, we generated an Escherichia coli strain producing β-apo-4′-carotenal, which was converted into neurosporaxanthin upon expression of carD. Moreover, the carD function was substantiated by its targeted disruption in a F. fujikuroi carotenoid-overproducing strain, which resulted in the loss of neurosporaxanthin and the accumulation of β-apo-4′-carotenal, its derivative β-apo-4′-carotenol, and minor amounts of other carotenoids. Intermediates accumulated in the ΔcarD mutant suggest that the reactions leading to neurosporaxanthin in Neurospora and Fusarium are different in their order. In contrast to ylo-1 in N. crassa, carD mRNA content is enhanced by light, but to a lesser extent than other enzymatic genes of the F. fujikuroi carotenoid pathway. Furthermore, carD mRNA levels were higher in carotenoid-overproducing mutants, supporting a functional role for CarD in F. fujikuroi carotenogenesis. With the genetic and biochemical characterization of CarD, the whole neurosporaxanthin biosynthetic pathway of F. fujikuroi has been established. Database The carD gene sequence has been deposited in the EMBL Data Bank under accession number FR850689

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 β-carotene cleavage oxygenases I and II catalyzing either a central or an excentric cleavage of β-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 (C20), β-apo-14′-carotenal (C22) and β-apo-13-carotenone (C18) from β-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.

Retinoids are C(20) apocarotenoids that have various important functions in metazoans. In addition, several findings suggest their occurrence in eubacteria, including cyanobacteria. It has been shown that the Synechocystis cytochrome P450 enzyme CYP120A1 is a retinoic acid-binding polypeptide. In this work, we determined the reaction catalyzed by CYP120A1 and investigated its substrate specificity in vitro. CYP120A1-containing microsomes generated in yeast converted all-trans-retinoic acid into a compound exhibiting higher polarity in HPLC analysis. Liquid chromatography-MS analysis suggested the introduction of a single hydroxyl group, and NMR analysis of the purified product revealed C16 or C17 as the reaction site. Incubations with cis-retinoic acids, retinal, 3(R)-OH-retinal, retinol, beta-apo-13-carotenone (C(18)) and beta-apo-14'-carotenal (C(22)) resulted in the formation of the corresponding hydroxyl derivatives, as suggested by HPLC and liquid chromatography-MS analyses. Comparisons of the relative product amounts revealed the highest conversion rate for all-trans-retinoic acid, followed by beta-apo-13-carotenone (C(18)). As shown by real-time RT-PCR, CYP120A1 is expressed under normal growth conditions and is slightly induced by high-intensity light. Our work provides the first enzymatic study of a cyanobacterial cytochrome P450, showing it to be the first nonanimal retinoic acid-metabolizing enzyme characterized so far. Moreover, the CYP120A1-catalyzed reaction represents a novel modification of retinoids.

Carotenoids are widespread terpenoid pigments with applications in the food and feed industries. Upon illumination, the gibberellin-producing fungus Fusarium fujikuroi (Gibberella fujikuroi mating population C) develops an orange pigmentation caused by an accumulation of the carboxylic apocarotenoid neurosporaxanthin. The synthesis of this xanthophyll includes five desaturation steps presumed to be catalysed by the carB-encoded phytoene desaturase. In this study, we identified a yellow mutant (SF21) by mutagenesis of a carotenoid-overproducing strain. HPLC analyses indicated a specific impairment in the ability of SF21-CarB to perform the fifth desaturation, as implied by the accumulation of gamma-carotene and beta-carotene, which arise through four-step desaturation. Sequencing of the SF21 carB allele revealed a single mutation resulting in an exchange of a residue conserved in other five-step desaturases. Targeted carB allele replacement proved that this single mutation is the cause of the SF21 carotenoid pattern. In support, expression of SF21 CarB in engineered carotene-producing Escherichia coli strains demonstrated its reduced ability to catalyse the fifth desaturation step on both monocyclic and acyclic substrates. Further mutagenesis of SF21 led to the isolation of two mutants, SF73 and SF98, showing low desaturase activities, which mediated only two desaturation steps, resulting in accumulation of the intermediate zeta-carotene at low levels. Both strains contained an additional mutation affecting a CarB domain tentatively associated with carotenoid binding. SF21 exhibited higher carotenoid amounts than its precursor strain or the SF73 and SF98 mutants, although carotenogenic mRNA levels were similar in the four strains.

Carotenoid cleavage products--apocarotenoids--include biologically active compounds, such as hormones, pigments and volatiles. Their biosynthesis is initiated by the oxidative cleavage of C-C double bonds in carotenoid backbones, leading to aldehydes and/or ketones. This step is catalyzed by carotenoid oxygenases, which constitute an ubiquitous enzyme family, including the group of plant carotenoid cleavage dioxygenases 1 (CCD1s), which mediates the formation of volatile C(13) ketones, such as beta-ionone, by cleaving the C9-C10 and C9'-C10' double bonds of cyclic and acyclic carotenoids. Recently, it was reported that plant CCD1s also act on the C5-C6/C5'-C6' double bonds of acyclic carotenes, leading to the volatile C(8) ketone 6-methyl-5-hepten-2-one. Using in vitro and in vivo assays, we show here that rice CCD1 converts lycopene into the three different volatiles, pseudoionone, 6-methyl-5-hepten-2-one, and geranial (C(10)), suggesting that the C7-C8/C7'-C8' double bonds of acyclic carotenoid ends constitute a novel cleavage site for the CCD1 plant subfamily. The results were confirmed by HPLC, LC-MS and GC-MS analyses, and further substantiated by in vitro incubations with the monocyclic carotenoid 3-OH-gamma-carotene and with linear synthetic substrates. Bicyclic carotenoids were cleaved, as reported for other plant CCD1s, at the C9-C10 and C9'-C10' double bonds. Our study reveals a novel source for the widely occurring plant volatile geranial, which is the cleavage of noncyclic ends of carotenoids.