1 3 Tomato carotenoid cleavage dioxygenases 1A and 1B: Relaxed 4 double bond specificity leads to a plenitude of dialdehydes, 5 mono-apocarotenoids and isoprenoid volatiles 6 7 8 Andrea Ilg a,1 Q1 , Mark Bruno a,1 , Peter Beyer a , Salim Al-Babili b,a,⇑ 9 a Albert-Ludwigs University of Freiburg, Faculty of Biology, Schaenzlestr. 1, D-79104 Freiburg, Germany 10 b Center for Desert Agriculture, BESE Division, King Abdullah University of Science and Technology (KAUST), 23955-6900 Thuwal, Saudi Arabia 11 12 14 article info 15 Article history: 16 Received 6 May 2014 17 Revised 19 June 2014 18 Accepted 19 June 2014 19 Available online xxxx 20 21 Q2 Keywords: 22 Apocarotenoids 23 Isoprenoids 24 Carotenoids 25 Carotenoid cleavage dioxygenase 26 Lycopene 27 Tomato 28 Plants 29 30 abstract 31 The biosynthetic processes leading to many of the isoprenoid volatiles released by tomato fruits are 32 still unknown, though previous reports suggested a clear correlation with the carotenoids contained 33 within the fruit. In this study, we investigated the activity of the tomato (Solanum lycopersicum) 34 carotenoid cleavage dioxygenase (SlCCD1B), which is highly expressed in fruits, and of its homolog 35 SlCCD1A. Using in vitro assays performed with purified recombinant enzymes and by analyzing 36 products formed by the two enzymes in carotene-accumulating Escherichia coli strains, we 37 demonstrate that SlCCD1A and, to a larger extent, SlCCD1B, have a very relaxed specificity for both 38 substrate and cleavage site, mediating the oxidative cleavage of cis- and all-trans-carotenoids as 39 well as of different apocarotenoids at many more double bonds than previously reported. This 40 activity gives rise to a plenitude of volatiles, mono-apocarotenoids and dialdehyde products, 41 including cis-pseudoionone, neral, geranial, and farnesylacetone. Our results provide a direct 42 evidence for a carotenoid origin of these compounds and point to CCD1s as the enzymes catalyzing 43 the formation of the vast majority of tomato isoprenoid volatiles, many of which are aroma 44 constituents. 45 Ó 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This 46 is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 47 48 49 50 1. Introduction 51 Carotenoids are lipophilic pigments produced by plants, photo- 52 synthetic prokaryotes and several heterotrophic bacteria and fungi. 53 Carotenoids play a vital role in photosynthesis, as pigments protect- 54 ing from photo-oxidation and contributing to the light-harvesting 55 process. Moreover, carotenoids serve as signals in the plant–animal 56 communication, since they are responsible for the color of many 57 fruits and flowers, e.g. tomato fruits and daffodil flowers, where 58 they frequently accumulate in chromoplasts [1–3]. Generally, ani- 59 mals lack the capability to synthesize carotenoids and, hence, they 60 need a dietary source for these pigments that act as antioxidants 61 and, more importantly, as precursors of vitamin A (retinol) and its 62 derivatives retinal and retinoic acid [4,5]. Furthermore, carotenoid 63 derived compounds other than retinoids, like b-apo-13-carotenone 64 and apolycopenals, are supposed to exert different biological activ- 65 ities in animal systems [6,7]. 66 Plant carotenoid biosynthesis occurs in plastids. The first step in 67 this pathway is the condensation of two molecules geranylgeranyl 68 diphosphate, which yields the first carotenoid 15-cis-phytoene. 69 This C 40 compound contains only three conjugated double bonds 70 and is, therefore, colorless. Desaturation and isomerization reac- 71 tions lead via specific cis-isomers of the intermediates phytofluene, 72 f-carotene, neurosporene and lycopene to all-trans-lycopene, the 73 red tomato fruit pigment with 11 conjugated double bonds. 74 Cyclization of all-trans-lycopene gives rise to b- or a-carotene that 75 can be hydroxylated to form zeaxanthin and lutein, respectively. 76 Zeaxanthin is the precursor of violaxanthin and neoxanthin (for 77 review, see [1–4,8]. 78 Due to the presence of a conjugated double bond system, carote- 79 noids are susceptible to oxidative cleavage that yields carbonyl 80 products, generally referred to as apocarotenoids. Some of the 81 apocarotenoids fulfill important environmental, physiological or 82 developmental functions, immediately or after being structurally 83 modified by other types of enzymes [9–12]. The group of apocarot- 84 enoids with known biological function includes retinoids, the http://dx.doi.org/10.1016/j.fob.2014.06.005 2211-5463/Ó 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Abbreviations: CCD, carotenoid cleavage dioxygenase ⇑ Corresponding author at: Division of Biological and Environmental Sciences and Engineering, Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology (KAUST), Building 2, Level 3, Room 3237, 23955-6900 Thuwal, Saudi Arabia. Tel.: +966 (12) 808 2762; fax: +966 (12) 802 0103. E-mail address: salim.babili@kaust.edu.sa (S. Al-Babili). 1 These authors contributed equally. FEBS Open Bio xxx (2014) xxx–xxx journal homepage: www.elsevier.com/locate/febsopenbio FOB 207 No. of Pages 10, Model 5G 1 July 2014 Please cite this article in press as: Ilg, A., et al. Tomato carotenoid cleavage dioxygenases 1A and 1B: Relaxed double bond specificity leads to a plenitude of dialdehydes, mono-apocarotenoids and isoprenoid volatiles. FEBS Open Bio (2014), http://dx.doi.org/10.1016/j.fob.2014.06.005