Biochemical and Biophysical Research Communications
Identification of a cyanobacterial aldehyde dehydrogenase that produces retinoic acid in vitro☆
Introduction
Retinoic acid is a key signalling molecule in healthy development and in differentiation of stem cells, albeit uncontrolled levels of retinoic acid can lead to mutagenesis [1]. In eukaryotes retinoic acid is made by oxidation of all-trans-retinal by retinal/aldehyde dehydrogenases (ALDH1A1, ALDH1A2, ALDH1A3, ALDH8) [2]. This step is preceded by the production of retinal from either retinol or beta-carotene (Fig. 1a). As yet, this canonical signalling pathway has only been characterised in animals.
Retinoic acid has more recently been identified in cyanobacteria both grown in culture and in blooms isolated in China, where it is thought to be responsible for the mutagenesis of frogs [[3], [4], [5], [6], [7], [8], [9]] Thus far, no mechanism for cyanobacterial retinoic acid production has been defined, beyond it being a potential degradation product of retinal [10].
Orthologues of carotenoid dioxygenases, used by humans to produce retinal from beta-carotene, have previously been characterised from the cyanobacteria Synechocystis PCC 6803 and Nostoc PCC 7107 [[11], [12], [13], [14]]. These orthologues are able to convert beta-carotene and apo-8-carotenal into all-trans-retinal. An orthologue of a cytochrome P450 enzyme known as CYP26 in animals and CYP120 in cyanobacteria has also been characterised and found to oxidise retinoic acid [[15], [16], [17]].
All-trans-retinal and the precursor beta-carotene have been identified in 24 species of cyanobacteria [18]. Production of all-trans-retinal, beta-carotene or retinoic acid in cyanobacteria occurs independently of whether the species is grown in culture or isolated from a bloom [4,18]. Therefore, given the conservation of genes involved in the synthetic pathway, one hypothesis is that the retinoic acid produced by cyanobacteria could be produced by the same biosynthetic pathway as in eukaryotes, potentially starting from beta-carotene (Fig. 1a).
Attempts have previously been made to identify a cyanobacterial aldehyde dehydrogenase that is able to convert all-trans-retinal to retinoic acid, bridging the gap between the previous characterised carotenoid dioxygenase and CYP120 [19]. This Synechocystis PCC 6803 slr0091 protein was able to convert long chain aldehydes into their respective acids but had no activity against retinal.
We have previously postulated that retinoic acid is not of animal origin as previously thought, but may be a product of bacterial origin and present in humans through a lateral gene transfer event of aldehyde dehydrogenase and CYP120 from cyanobacteria to animals [20]. Previously, lateral gene transfers between cyanobacteria and animals, in particular of cyclooxygenases [21], calpains [22] and WD40 domains have been suggested [23], indicating that these events are likely to have shaped processes across the animal kingdom.
Our phylogenetic analysis identified orthologues of human ALDH1A1 in cyanobacteria that have a very high sequence conservation (>60%) and retain the catalytic GQCC motif. Herein we characterise in vitro a cyanobacterial ALDH and demonstrate that it can convert retinal to retinoic acid.
Section snippets
Results
A good model for the study of retinoic acid production in cyanobacteria would have orthologues of all the proteins in the putative pathway. Therefore, we searched the NCBI database of non-redundant protein sequences for a cyanobacterium that contains orthologues of human ALDH1A2 (BAA34785.1), beta-carotene monooxygenase (AAI26211.1) and cytochrome P450 CYP26A1 (AAB88881.1). One of 65 cyanobacteria identified that possess all three orthologues (Table S1), was Chlorogloeopsis fritschii PCC 6912.
Discussion
We have identified and characterised for the first time a cyanobacterial ALDH that can convert all-trans-retinal to retinoic acid. Blooms producing retinoic acid are proving toxic to local flora and fauna, so any further insight into the mechanism of this production is crucial. This is a potentially important step in elucidation of the biosynthesis of retinoic acid in cyanobacteria, though further experimentation is required to determine if this enzyme is in fact employed for this purpose in
Phylogenetic analyses
Aldehyde dehydrogenase sequences were chosen from Refs. [20,24], along with the Bacillus subtilis ALDH from Ref. [25] and slr0091 from Synechocystis PCC 6803 [19]. Amino acid sequences were aligned using MUSCLE [37] in MEGA7 [38]. Phylogeny was inferred by Maximum Likelihood using the LG model [39] in MEGA7 with 1000 bootstrap replicates. Full details of sequences are in Supplementary Information Table 2.
Cloning, expression and purification cfALDH
A cyanobacterial ALDH from Chlorogloeopsis fritschii PCC 6912 was identified by BLASTp
Acknowledgements
We thank Dr David Adams at the University of Leeds for providing us with Chlorogloeopsis fritschii PCC 6912. This phylogenetic work was undertaken on MARC1, part of the High Performance Computing and LIDA facilities at the University of Leeds, UK.
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Paul Taylor is a Director of Tangent Reprofiling Limited. Other authors have no competing financial interests.