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Clinical Science (2000) 28, (806–810) (Printed in Great Britain)
Molecular evolution of lycopene cyclases involved in the formation of carotenoids with ionone end groups
P. Krubasik and G. Sandmann1
Biosynthesis Group, Botanical Institute, J. W. Goethe Universität, P.O. Box 111930, D-60054 Frankfurt, Germany
Key words: capsanthin/capsorubin synthase, fungal lycopene b-cyclase, heterodimeric lycopene cyclase, lycopene-e cyclase, lycopene cyclase/phytoene synthase fusion. Abbreviation used: Ccs, capsanthin capsorubin synthase. 1To whom correspondence should be addressed (e-mail Sandmann@em.uni-frankfurt.de). Abstract A survey is given of the lycopene cyclase genes present in bacteria, fungi and plants where two completely unrelated types exist. One is the classical monomeric bacterial b-cyclase gene, crtY, which may be an ancestor of crtL, the gene for a b-cyclase in cyanobacteria. From crtL a line of evolution can be drawn to plant b- and e-cyclase genes and to the gene of capsanthin/capsorubin synthase. In Gram-positive bacteria two genes crtYc and crtYd are present. They encode two proteins which have to interact as a heterodimer for lycopene b-cyclization. From this type of lycopene cyclase gene the fungal lycopene cyclase/phytoene synthase fusion gene evolved. Introduction Carotenoids are widely distributed in Nature and more than 600 different structures are known. Plants, several bacteria and some fungi are able to synthesize carotenoids. Even though the end products of carotenoid biosynthesis can be very diverse, a general common pathway leading to the formation of cyclic b-carotene can be observed in many prokaryotic and eukaryotic organisms. The synthesis of this C40 carotene starts by the condensation of two molecules of geranylgeranyl pyrophosphate followed by four desaturation steps. Then, the ends of the resulting acyclic lycopene may be cyclized to b-ionone, e-ionone or g-ionone rings. The enzymes involved in these cyclization reactions are lycopene cyclases. The genes of lycopene cyclases have been cloned from many bacteria, from fungi and from plants. The classical monomeric lycopene cyclases of plants and many bacteria The first lycopene cyclase gene, crtY, was cloned from the eubacterium Erwinia uredovora [1]. Several other very similar crtY genes are now available from other non-photosynthetic bacteria. So far the only cyanobacterial lycopene cyclase gene (crtL) is known from Synechococcus sp. PCC7942 [2]. Its similarity to the bacterial crtY-type cyclases is rather low. Nevertheless, distinct conserved patterns in the amino acid sequence can be observed in crtY, crtL and the lycopene cyclase genes from plants (Figure 1). These are one hypothetical dinucleotide-binding region with similarities to the one found in phytoene desaturases [3] (Figure 1, box A) and four further motifs with unknown function [4] (Figure 1, boxes B–E). A common phylogenetic origin of crtY, crtL and lycopene cyclase genes from plants is therefore assumed [5]. The amino acid sequence of lycopene cyclases from plants and cyanobacteria are closely related, as indicated by the phylogenetic tree (Figure 2A). All these lycopene cyclases are polypeptides with around 400 amino acids and a molecular mass of 43 kDa [6], and the enzymes from plants have an additional N-terminal transit sequence of 100 amino acids. The formation of b-ionone and e-ionone rings in plants is catalysed by two different enzymes, b-cyclase and e-cyclase. Both enzymes show high similarities in their amino acid sequence and it is very likely that they evolved from the same ancestor. Another member of this protein family of plant lycopene cyclases is the capsanthin capsorubin synthase (Ccs) from Capsicum annuum [7]. This enzyme converts antheraxanthin or violaxanthin into capsanthin or capsorubin by a mechanism similar to lycopene cyclization [8]. In addition, Ccs can also convert lycopene into b-carotene and the amino acid sequences of Ccs and lycopene cyclases are very similar. Therefore it was proposed that ccs evolved from a duplicated lycopene cyclase gene.The new heterodimeric lycopene cyclases A second type of lycopene cyclase was recently described for the Gram-positive coryneform bacterium Brevibacterium linens [9]. In this new type of lycopene cyclase the two different genes crtYc and crtYd code for two small polypeptides of 125 and 107 amino acids, respectively. Only the combined products of both genes are able to convert lycopene into b-carotene. Thus the functionalenzyme is referred to as heterodimeric lycopene cyclase. The genes of these lycopene cyclases show no sequence similarities with any known genes or with any of the conserved regions of the classical lycopene cyclases. The same type of cyclases has been functionally identified in the carotenoid gene cluster of Mycobacterium aurum [10] and similar sequences were found near a functionally identified phytoene synthase gene in the genome of Mycobacterium marinum [11]. Genome sequencing data also reveals a similar lycopene cyclase for Mycobacterium avium (http://www.tigr.org, database m-avium, contig 89). As shown in the alignments of Figure 2(B), these genes are highly homologous and share several conserved regions. None of them could be found in the crtY genes. All species possessing the crtYc and crtYd genes belong to the order Actinomycetales, a group of Gram-positive bacteria with a high GC content in their DNA. Surprisingly, in Streptomyces griseus, also belonging to the Actinomycetales, a lycopene cyclase of the classical CrtY-type has been functionally identified [12]. The sequencing project of Streptomyces coelicolor (http://www.sanger.ac.uk./projects/S-coelicolor, cosmid StJ12) also revealed the presence of a classical lycopene cyclase. In summary, the carotenoid-producing Actinomycetales seem to have the new type of heterodimeric lycopene cyclase, with the exception of Streptomyces species, which have the classical CrtY-type lycopene cyclase. Interestingly, the carotenoid gene cluster of the Gram-negative bacterium Myxococcus xanthus also has genes with similarities to crtYc and crtYd from B. linens [13]. Even though a function could not be assigned to these two genes, it can be expected that they encode the lycopene cyclase of this organism, which synthesizes cyclic carotenoids. The bifunctional fungal lycopene cyclases The first lycopene cyclase gene from a fungus was isolated and identified from the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous [14]. Its cDNA encodes a bifunctional lycopene cyclase/phytoene synthase of 673 amino acids. This gene product has two catalytic activities in carotenoid biosynthesis as it converts geranylgeranyl phosphate into phytoene as well as lycopene into b-carotene. The deduced amino acid sequence of this cDNA has similarities in its C-terminal part to phytoene synthases from other organisms and has N-terminal similarities to the new heterodimeric lycopene cyclases CrtYc and CrtYd from B. linens (Figure 2B). Owing to the bifunctionality of its gene product, the gene was called crtYB. By truncation of the crtYB cDNA it could be demonstrated that its phytoene synthase and lycopene cyclase domains are localized in the regions of the polypeptide with sequence similarities to phytoene synthases and the new lycopene cyclases, respectively. The gene product of al-2 from Neurospora crassa, which has been shown to be involved in phytoene synthesis of this ascomycetous fungus [15], has a high overall sequence similarity to CrtYB from X. dendrorhous. Like CrtYB the al-2 gene product is not only a phytoene synthase but also exhibits lycopene cyclase activity (P. Krubasik and G. Sandmann, unpublished work). By database searches a further sequence from the zygomycetous fungus Mucor circinelloides with similarities to CrtYB was found and it can be assumed that it also encodes a bifunctional lycopene cyclase/phytoene synthase. Moreover, based on classical genetic studies, a common translation or a fusion of lycopene cyclase and phytoene synthase has been predicted for M. circinelloides [16]. Lycopene cyclase genes from all big groups of phylogenetically related fungi, basidiomycetes, ascomycetes, and zygomycetes, were found. As the first two groups developed from forms of the latter, it can be supposed that this unique type of lycopene cyclase/phytoene synthase must have been acquired relatively early in the evolution of fungi. The bifunctional lycopene cyclase might have originated from the fusion of two crtYc- and crtYd-type genes, similar to those in B. linens, and from a phytoene synthase. The fusion of crtYc, crtYd and the phytoene synthase gene might have occurred by recombinant processes. An example of how a carotenogenic fusion gene could originate by chromosomal rearrangement was recently given [17]. In a Rubrivivax gelatinosous strain with an inactivated crtB gene, which therefore is devoid of carotenoids, illegitimate recombination of a new functional chimaeric crtB gene was forced by photo-oxidative stress. Other types of lycopene cyclase At least one further type of lycopene cyclase has to be expected, as sequence comparisons of the known lycopene cyclases with the genome of the fully sequenced cyanobacterium Synechocystis sp. PCC6804 [18] did not result in a significant match with any known lycopene cyclase. A lycopene cyclase gene must exist in this organism because b-carotene, which is the product of lycopene cyclization, was found in this cyanobacterium [19]. The genomic sequence data of one further completely sequenced cyanobacterium, Anabaena sp. PCC7120 (http://www.kazusa.or.jp/cyano/anabaena), did also lack similarities to the sequences of known lycopene cyclase genes. Conclusion on the phylogeny of lycopene cyclase genes Two completely unrelated lycopene b-cyclases evolved independently in bacteria. The heterodimeric lycopene b-cyclase encoded by the genes crtYc and crtYd dominates among Gram-positive bacteria. From these genes the fungal lycopene b-cyclase/phytoene synthase fusion gene crtYB derived. The completely unrelated crtY of a monomeric b-cyclase, identified first in a Gram-negative enterobacterium, may be an ancestor of a cyanobacterial and prochlorophyte lycopene b-cyclase gene crtL. From this gene, a line of evolution can be drawn to the corresponding lcy-b genes in chlorophytes and higher plants. The genes encoding lycopene e-cyclase, lcy-e, and Ccs, ccs, from plants are highly homologous with the b-cyclase genes and may originate from gene duplications. References 1 Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K. and Harashima, K. (1990) J. Bacteriol. 172, 6704–6712 2 Cunningham, F. X., Sun, Z., Chamovitz, D., Hirschberg, J. and Gantt, E. (1994) Plant Cell 6, 1107–1121 3 Armstrong, G. A., Hundle, B. S. and Hearst, J. E. (1993) Methods Enzymol. 214, 297–311 4 Cunningham, Jr, F. X., Pogson, B., Sun, Z., McDonald, K. A., DellaPenna, D. and Gantt, E. (1996) Plant Cell 8, 1613–1626 5 Hugueney, P., Badillo, A., Cehn, H.-C., Klein, A., Hirschberg, J., Camara, B. and Kuntz, M. (1995) Plant J. 8, 417–424 6 Schnurr, G., Misawa, N. and Sandmann, G. (1996) Biochem. J. 315, 869–874 7 Bouvier, F., Hugueney, P., d'Harlingue, A., Kuntz, M. and Camara, B. (1994) Plant J. 6, 45–54 8 Bouvier, F., d'Harlingue, A. and Camara, B. (1997) Arch. Biochem. Biophys. 346, 53–64 9 Krubasik, P. and Sandmann, G. (2000) Mol. Gen. Genet. 263, 423–432 10 Viveiros, M., Krubasik, P., Houssaini-Iraqui, M., Sandmann, G. and David, H. L. 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(1985) Phytochemistry 24, 2919–2922 Received 22 June 2000 Copyright 2000 Biochemical Society |
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