Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid- dependent axial patterning in metazoans Tiago J. P. Sobreiraa,1, Ferdinand Marlétazb,1, Marcos Simões-Costac,d,1, Deborah Schechtmane, Alexandre C. Pereiraa, Frédéric Brunetb, Sarah Sweeneyf, Ariel Panig,h, Jochanan Aronowiczh, Christopher J. Loweh, Bradley Davidsonf, Vincent Laudetb, Marianne Bronneri, Paulo S. L. de Oliveiraa, Michael Schubertb,2,3, and José Xavier-Netoc,2,3 aLaboratório de Genética e Cardiologia Molecular, Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, 05403-000, São Paulo-SP, Brazil; bInstitut de Génomique Fonctionnelle de Lyon, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France; cLaboratório Nacional de Biociências, Campus do Laboratório Nacional de Luz Síncrotron, 13083-970, Campinas-SP, Brazil; dDepartamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, University of São Paulo, 05508-900, São Paulo-SP, Brazil; eDepartamento de Bioquímica, Instituto de Química da Universidade de São Paulo, 05508-900, São Paulo-SP, Brazil; fDepartment of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85724; gCommittee on Evolutionary Biology, University of Chicago, Chicago, IL 60637; hHopkins Marine Station, Department of Biology, Stanford University, Pacific Grove, CA 93950; and iDivision of Biology 139-74, California Institute of Technology, Pasadena, CA 91125 Edited* by John Gerhart, University of California, Berkeley, CA, and approved November 10, 2010 (received for review August 17, 2010) Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and Second, ALDHs are among the best-characterized proteins, and process the vitamin A-derived retinaldehyde into retinoic acid (RA), their structure and substrate profiles have been determined with a small diffusible molecule and a pivotal chordate morphogen. In exquisite precision (9–15). Thus, structural modeling of these this study, we combine phylogenetic, structural, genomic, and de- proteins can be used to study the evolution of substrate specificity velopmental gene expression analyses to examine the evolutionary without extensive biochemical analyses (16–20). origins of ALDH substrate preference. Structural modeling reveals ALDH1 and ALDH2 enzymes share a high degree of sequence that processing of small aldehydes, such as acetaldehyde, by identity, indicating a very close phylogenetic relationship (3). Pi- ALDH2, versus large aldehydes, including retinaldehyde, by oneer observations by Moore et al. on human ALDH2 and sheep EVOLUTION ALDH1A is associated with small versus large substrate entry chan- ALDH1 (17) suggested that their respective abilities to detoxify nels (SECs), respectively. Moreover, we show that metazoan small aldehydes and to process large aldehydes are correlated with ALDH1s and ALDH2s are members of a single ALDH1/2 clade and the size and shape of their substrate entry channels (SECs), the that during evolution, eukaryote ALDH1/2s often switched be- intramolecular cavities that direct aldehydes to the catalytic sites of ALDH enzymes. Human ALDH2 displays a narrow SEC with tween large and small SECs after gene duplication, transforming a constricted entrance, whereas sheep ALDH1A1 exhibits a large constricted channels into wide opened ones and vice versa. Ances- SEC with a broad opening (17, 18). Thus, SEC topology influences tral sequence reconstructions suggest that during the evolutionary ALDH1/2 substrate preference. For example, although reti- emergence of RA signaling, the ancestral, narrow-channeled meta- naldehyde is a good substrate for vertebrate ALDH1s and acet- zoan ALDH1/2 gave rise to large ALDH1 channels capable of accom- aldehyde is a natural substrate of ALDH2s, ALDH2s cannot modating bulky aldehydes, such as retinaldehyde, supporting the process retinaldehyde and ALDH1s process acetaldehyde only view that retinoid-dependent signaling arose from ancestral cellu- extremely inefficiently (16, 17–22). lar detoxification mechanisms. Our analyses also indicate that, on To understand the evolutionary origins of the substrate pref- a more restricted evolutionary scale, ALDH1 duplicates from inver- erences of ALDH1 and ALDH2 enzymes, as well as to illuminate tebrate chordates (amphioxus and ascidian tunicates) underwent how signaling and protective functions are connected to these switches to smaller and narrower SECs. When combined with alter- different enzyme activities, we used an integrated approach that ations in gene expression, these switches led to neofunctionaliza- combined genomic, phylogenetic, and structural analyses. The tion from ALDH1-like roles in embryonic patterning to systemic, resulting comprehensive data set was complemented with in- ALDH2-like roles, suggesting functional shifts from signaling to formation on developmental gene expression of ALDH1/2s in the detoxification. cephalochordate amphioxus (Branchiostoma floridae) and the ascidian tunicate Ciona intestinalis. These two invertebrate chor- Aldehyde dehydrogenase phylogeny | Branchiostoma floridae | Ciona date models possess functional RA signaling cascades and are intestinalis versus Ciona savignyi | evolution of retinoic acid signaling | pivotal models for understanding vertebrate origins from both origins of morphogen-dependent signaling a genomic and a developmental perspective (4, 23–26). Together, this work provides support for the hypothesis that some in- tercellular signaling mechanisms evolved from cellular de- n animal development, major signaling pathways are controlled fi Iby morphogens, diffusible molecules whose evolutionary origins toxi cation pathways. are difficult to assess. Aldehyde dehydrogenase (ALDH) enzymes are attractive subjects to study the evolution of morphogen sig- naling for two main reasons. First, in addition to their acknowl- Author contributions: M.S. and J.X.-N. designed research; T.J.P.S., F.M., M.S.-C., D.S., F.B., S.S., A.P., J.A., C.J.L., B.D., P.S.L.d.O., M.S., and J.X.-N. performed research; D.S., C.J.L., B.D., edged role in protecting animals by catabolizing reactive biogenic M.B., and P.S.L.d.O. contributed new reagents/analytic tools; T.J.P.S., F.M., M.S.-C., D.S., and xenobiotic aldehydes, some ALDHs also synthesize signaling A.C.P., F.B., S.S., A.P., J.A., C.J.L., B.D., V.L., P.S.L.d.O., M.S., and J.X.-N. analyzed data; and molecules (1–3). Prime examples for these two ALDH enzyme T.J.P.S., F.M., D.S., B.D., V.L., M.B., P.S.L.d.O., M.S., and J.X.-N. wrote the paper. roles are the ALDH2s, which degrade small toxic aldehydes, The authors declare no conflict of interest. such as the acetaldehyde derived from ethanol metabolism (1, 2), *This Direct Submission article had a prearranged editor. and the ALDH1s, which process larger aldehydes, including ret- 1T.J.P.S., F.M., and M.S.-C. contributed equally to this work. inaldehyde, a vitamin A-derived precursor of the morphogen 2M.S. and J.X.-N. contributed equally to this work. retinoic acid (RA). RA plays a critical role during embryonic 3To whom correspondence may be addressed. E-mail: [email protected] or development of chordates (i.e., amphioxus, tunicates, and verte- [email protected]. brates) and has been suggested to have already been involved in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. patterning the last common ancestor of bilaterian animals (4–8). 1073/pnas.1011223108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1011223108 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Results we performed large-scale phylogenetic analyses of the ALDH SEC Volumes Distinguish Vertebrate ALDH1s from ALDH2s. To test superfamily (Fig. 1, Figs. S1 and S2, Dataset S1, and Dataset S2). whether SEC differences between sheep ALDH1 and human Contrary to traditional views, we found that metazoan ALDH1s ALDH2 reflect fundamental evolutionary differences between and ALDH2s do not form independent families but are members these enzymes, we used the crystal structures of these proteins to of a single, well-supported ALDH1/2 clade, with ALDH2s create 3D structural models of ALDH1/2s. These models were forming a distinct group nested within this ALDH1/2 clade (Fig. 1 then analyzed to determine molecular parameters, such as SEC and Figs. S1 and S2). In contrast to ALDH2s, ALDH1s un- volume, which are implicated in substrate preference (17). Our derwent multiple lineage-specific duplications. For example, the dataset shows that ALDH1s generally display larger channel genomes of amphioxus (B. floridae) and the ascidian tunicates volumes than ALDH2s (589 ± 59 Å3 for ALDH1s versus 403 ± C. intestinalis and C. savignyi contain, respectively, six, four, and 53 Å3 for ALDH2s, mean ± SD, P < 0.001) (Dataset S1 and two ALDH1 duplicates, whereas in the hemichordate Saccoglossus Dataset S2). Therefore, channel volume represents a funda- kowalevskii, there are five ALDH1 genes (Fig. 1, Figs. S1 and S2, mental difference between ALDH1 and ALDH2, reflecting Dataset S1, and Dataset S2) (27). conserved structural requirements associated with processing of Analyses of channel size distribution in eukaryote ALDH1s large and small aldehydes, respectively. and ALDH2s indicate that SEC variation can be subdivided into small (<420 Å3), medium (420–508 Å3), and large channels Because it is likely that the overall geometry of the SEC, rather 3 than its volume alone, determines ALDH1/2 specificities, we (>508 Å )(Fig. S3,