SEC Volumes Distinguish Vertebrate ALDH1s from ALDH2s.

To test whether SEC differences between sheep ALDH1 and human ALDH2 reflect fundamental evolutionary differences between these enzymes, we used the crystal structures of these proteins to create 3D structural models of ALDH1/2s. These models were then analyzed to determine molecular parameters, such as SEC volume, which are implicated in substrate preference (17). Our dataset shows that ALDH1s generally display larger channel volumes than ALDH2s (589 ± 59 ų for ALDH1s versus 403 ± 53 ų for ALDH2s, mean ± SD, P < 0.001) (Dataset S1 and Dataset S2). Therefore, channel volume represents a fundamental difference between ALDH1 and ALDH2, reflecting conserved structural requirements associated with processing of large and small aldehydes, respectively.

Because it is likely that the overall geometry of the SEC, rather than its volume alone, determines ALDH1/2 specificities, we looked for further mechanistic clues to the evolution of substrate preference in ALDH1/2 sequences that diverge between the biochemically well-characterized vertebrate ALDH1s and ALDH2s. We hence compiled a list of 34 amino acid sequence signatures distinguishing the large-channeled ALDH1As (known to synthesize RA) from the narrow-channeled ALDH2s (with well-characterized roles in detoxification of small toxic aldehydes). Six signatures mapped to a subset of the 27 amino acids that form the SEC (Table S1). Of those signatures, only three signatures are positioned at critical locations at the ALDH1/2 channel, suggesting that they are implicated in determining ALDH1 and ALDH2 channel volumes/functions. The remaining signatures marked residues at oligomerization domains, surface loops or inside the molecule, which, after careful examination, did not suggest immediate functional correlates (Table S1).

The first of the three SEC signatures includes amino acid 124 at the entrance (“mouth”) of the ALDH1/2 channel (17). In human ALDH2, a bulky Met124 (124 ų van der Waals volume) protrudes into the channel (Table S1), allowing access of only small aldehydes, whereas in sheep ALDH1A1, a small, unobtrusive Gly124 (48 ų) allows entry of large aldehydes (17, 18). Thus, the first amino acid signature performs a size selection function. The second channel signature includes amino acid 459 at the proximal third (“neck”) of the ALDH1/2 SEC (Table S1) (17). In vertebrate ALDH2s, this amino acid is a large Phe459 (135 ų). In contrast, vertebrate ALDH1s typically display the smaller Val (93 ų) or Leu (124 ų) (Table S1). The third signature corresponds to aa303, close to the catalytic Cys302 (86 ų) at the end of the channel (“bottom”) (17). In vertebrate ALDH2s, this amino acid is Cys303, whereas vertebrate ALDH1s typically display Thr303 (93 ų), Ile303 (124 ų), or Val303 (Table S1).

To understand the roles of the amino acid signatures in substrate interaction, we performed docking studies on human ALDH2 and sheep ALDH1 with small aldehydes (Movie S1, Movie S2, and Movie S3). In the ALDH2 channel, acetaldehyde is kept close to the γ-sulfur of the catalytic Cys302 (Movie S1), whereas the ALDH1 channel does not favor this close association between the aldehyde substrate and Cys302, neither for acetaldehyde (Movie S2), nor for formaldehyde (Movie S3). Analysis of the structural motifs involved in substrate retention close to the ALDH catalytic site indicates that, in ALDH2, Cys302 and Phe465 keep acetaldehyde close to the catalytic γ-sulfur and that Phe465 is latched in position by Phe459, which, in turn, is fixed by Cys303. In ALDH1, this mechanism is not present, because the interaction surfaces between Cys303 and Phe459 are missing, due to their substitution by Ile303 and Val459, respectively (Movie S2). Thus, neck and bottom signatures keep the aldehyde moiety of small substrates close to the catalytic Cys302 in ALDH2s, consistent with a structural specialization of narrow-channeled ALDHs to process small aldehydes.

Switches from Small to Large ALDH1/2 Substrate Entry Channels Operated at the Origin of RA Signaling.

To understand the evolution of affinities for small and large aldehydes in ALDH2 and ALDH1, we performed large-scale phylogenetic analyses of the ALDH superfamily (Fig. 1, Figs. S1 and S2, Dataset S1, and Dataset S2). Contrary to traditional views, we found that metazoan ALDH1s and ALDH2s do not form independent families but are members of a single, well-supported ALDH1/2 clade, with ALDH2s forming a distinct group nested within this ALDH1/2 clade (Fig. 1 and Figs. S1 and S2). In contrast to ALDH2s, ALDH1s underwent multiple lineage-specific duplications. For example, the genomes of amphioxus (B. floridae) and the ascidian tunicates C. intestinalis and C. savignyi contain, respectively, six, four, and two ALDH1 duplicates, whereas in the hemichordate Saccoglossus kowalevskii, there are five ALDH1 genes (Fig. 1, Figs. S1 and S2, Dataset S1, and Dataset S2) (27).

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Fig. 1.

Phylogeny of ALDH1/2s, ALDH1Ls, and ALDH8s. The overall topology with the ALDH8s used as outgroup is shown in A. The boxed area in A highlights the metazoan ALDH1/2 clade, which is depicted in B. Nodes with significant posterior probabilities (>0.95) are highlighted with icons. The actual values are given in Fig. S2. Channel size categories are shown diagrammatically for ALDH1/2s and ALDH1Ls with sufficient sequence information. ALDH8s are too divergent for these calculations. At, Arabidopsis thaliana; Bf, Branchiostoma floridae; Ct, Capitella teleta; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Cs, Ciona savignyi; Dm, Drosophila melanogaster; Hs, Homo sapiens; Lg, Lottia gigantea; Mm, Mus musculus; Nv, Nematostella vectensis; Nt, Nicotiana tabacum; Os, Oryza sativa; Pc, Phanerochaete chrysosporium; Pb, Phycomyces blakesleeanus; Pt, Populus trichocarpa; Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae; Sk, Saccoglossus kowalevskii; Sp, Strongylocentrotus purpuratus; Ssp, Schizosaccharomyces pombe; Zm, Zea mays.

Analyses of channel size distribution in eukaryote ALDH1s and ALDH2s indicate that SEC variation can be subdivided into small (<420 ų), medium (420–508 ų), and large channels (>508 ų) (Fig. S3, Dataset S1, and Dataset S2). In metazoans, small channels dominate in ALDH2s, whereas large channels preponderate in ALDH1s (Fig. 1). In plants, there are also two major ALDH1/2 groups, one with small and medium channels and another one with predominantly large channels (345 ± 47 ų versus 561 ± 78 ų, P < 0.05), suggesting that a single ALDH1/2 ancestor duplicated early in plant evolution, giving rise to two distinct functional classes (28). Fungi experienced a distinct diversification pattern with various fungal lineages independently duplicating a single ALDH1/2 ancestor (29). These duplicates subsequently underwent SEC alterations, leading to fungal ALDH1/2 enzymes with small, medium or large SECs (Fig. 1).

To understand how small, medium, and large SEC volumes arose during evolution of the ALDH1/2 clade, we reconstructed ancestral sequences at selected nodes of the ALDH1/2 phylogeny by using the maximum likelihood method (Table S2). For example, the reconstructed ancestral eukaryote ALDH1/2 enzyme displays a small SEC, in which a bulky Met124 blocks the channel mouth, Phe459 constricts the channel neck, and Cys303 occupies the channel bottom, similar to modern metazoan ALDH2 SECs (Table S2). This reconstruction indicates that the vertebrate ALDH2 SEC signatures that distinguish them from vertebrate ALDH1s are ancestral, rather than derived, reflecting ancient structural adaptation to process small aldehydes. Table S2 also shows that the emergence of large-channeled ALDH1 enzymes was accompanied by substitution of an ancestral bulky Met124 at the SEC mouth by small amino acids, such as Gly and Ala. This observation indicates that the wide open channels of ALDH1 enzymes, which process large aldehydes, such as retinaldehyde, evolved from a background of small, constricted channels similar to those displayed by vertebrate ALDH2s, which process small, toxic aldehydes.

ALDH1 Duplication and Divergence Switched Large into Narrow Substrate Entry Channels in Invertebrate Chordates.

Curiously, the three SEC signatures that efficiently discriminate large-channeled vertebrate ALDH1s from narrow-channeled vertebrate ALDH2s do not distinguish their invertebrate chordate counterparts, because some invertebrate chordate ALDH1s display channel signatures typical for vertebrate ALDH2s (Table S1). To determine how these SEC variations evolved in the framework of the multiple, lineage-specific ALDH1 duplications of invertebrate chordates, we focused on the cephalochordate amphioxus, whose genome encodes six ALDH1s and a single ALDH2 (27), and on two closely related ascidian tunicates: C. intestinalis with four ALDH1s and a single ALDH2, and C. savignyi with two ALDH1s and a single ALDH2.

Because the first ALDH2 signature at the channel mouth probably selects for smaller substrates, we hypothesized that bulky and small residues may be similarly present in invertebrate chordate ALDH2s and ALDH1s, respectively. Accordingly, a bulky Leu124 protrudes into the channel mouth of amphioxus, C. intestinalis, and C. savignyi ALDH2s (Fig. 2, and Figs. S4 and S5). Amphioxus ALDH1s are heterogeneous in that a small Gly124 is embedded into the channel border without constricting the channel in amphioxus ALDH1a and ALDH1d, whereas the larger Glu124 (109 ų) or Ser124 (73 ų) partially obstruct the channel in the other four amphioxus duplicates, leaving insufficient space to accommodate the β-ionone moiety of retinaldehyde (Fig. 2). The ALDH1s from C. intestinalis and C. savignyi are also heterogeneous: C. intestinalis ALDH1a and ALDH1d, as well as C. savignyi ALDH1a, display the small Gly and C. savignyi ALDH1b, the small Ala (67 ų) at position 124, which do not obstruct the channel entrance, whereas C. intestinalis ALDH1b and ALDH1c, respectively, display the larger Ser124 and Ile124, which interfere with retinaldehyde accommodation in the ALDH1 channel (Fig. 2, and Figs. S4 and S5).

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Fig. 2.

Amphioxus ALDH1/2 duplicates. Phylogeny (A), channel structure (B), and developmental expression (C). Amino acid signatures of the substrate entry channel at positions 124 (the mouth), 459 (the neck), and 303 (the bottom) are indicated. For the expression analyses, neurulae (12 h) and late embryos (24 h) are shown. (Scale bars: 50 μm.)

In the ALDH2s from amphioxus, C. intestinalis, and C. savignyi, the amino acid at the second channel signature is Phe459, which constricts the channel neck with its large aromatic ring. As in vertebrate ALDH1s, in some amphioxus ALDH1s, the bulky Phe459 is substituted by smaller amino acids, such as Ile459 in ALDH1a, ALDH1b, and ALDH1d, Thr459 (93 ų) in ALDH1c or Gly459 in ALDH1e and ALDH1f. In ascidian tunicates, only C. intestinalis ALDH1d displays a Leu459, whereas all other ALDH1s display bulky amino acids, such as Phe and Met at position 459 (Figs. S4 and S5), similar to vertebrate ALDH2s.

As in vertebrates, in amphioxus, C. intestinalis, and C. savignyi, the amino acids of the third ALDH2 signature at the channel bottom is Cys303. Amphioxus ALDH1s are heterogeneous in that ALDH1a and ALDH1d display the vertebrate ALDH1 pattern (Thr303 and Ile303, respectively), whereas all other amphioxus ALDH1s display the vertebrate ALDH2 pattern (Fig. 2). In ascidian tunicates, only C. intestinalis and C. savignyi ALDH1a display the vertebrate ALDH1 pattern with Thr303, whereas other ALDH1s show the vertebrate ALDH2 pattern (Figs. S4 and S5). Thus, after lineage-specific duplication, some ALDH1 enzymes of invertebrate chordates incorporated amino acids and/or structural motifs similar to those of the vertebrate ALDH2 channel, shifting from a large, wide open configuration to constricted SEC topologies.

Synteny analyses in amphioxus and C. intestinalis suggest that the ALDH1a genes from both species are the sister groups to the other cephalochordate and ascidian tunicate ALDH1s and that the amphioxus duplicates ALDH1b, ALDH1c, ALDH1d, ALDH1e, and ALDH1f and C. intestinalis ALDH1b, ALDH1c, and ALDH1d evolved by duplication from an ALDH1a-like ancestor (Fig. S6). Moreover, reconstructions of ancestral SEC signatures and channel structures indicate that amphioxus and C. intestinalis ALDH1 ancestors displayed large SECs, structurally consistent with the capacity to process retinaldehyde into RA (Figs. 1 and 2, and Figs. S2 and S4). The reconstructed amphioxus ALDH1 ancestor already displayed a typical ALDH1 SEC with a 512 ų–wide opening lined by Gly124, an unobstructed neck flanked by Val459, and a bottom Cys303. The reconstructed C. intestinalis ALDH1 ancestor exhibits a large, 540 ų SEC, but, curiously, with ancestral Met124, Phe459, and Cys303 SEC signatures, suggesting that large ALDH1 channels emerged independently and by different mechanisms in cephalochordates and ascidian tunicates.

Invertebrate Chordate ALDH1 Channel Switch Is Associated with Transitions Between Restricted and Pleiotropic Expression.

Our next goal was to understand the specific developmental contexts in which the invertebrate chordate ALDH1/2s are deployed. We hence assessed developmental expression of the large-channeled ALDH1s structurally capable of accommodating retinaldehyde for RA synthesis, the narrow-channeled ALDH2 genes adapted for small aldehyde detoxification, and the divergent, narrow-channeled ALDH1s.

The ALDH1a and ALDH1d genes of amphioxus and C. intestinalis encode enzymes with large and unobstructed SECs. Amphioxus ALDH1a is expressed caudally close to the developing tail bud with a sharp anterior boundary in the neurula (at 12 h) (Fig. 2). In C. intestinalis, ALDH1a is expressed in a sharp, posterior mesodermal domain in the early embryo (at 5–8 h) (Fig. S4) (30). At later embryonic stages, ALDH1a is detectable in a distinct domain in the posterior gut endoderm of the amphioxus late embryo (at 24 h) and in the posterior trunk of C. intestinalis (at 10–12 h). In amphioxus, expression of ALDH1d overlaps that of ALDH1a at the neurula stage, but diverges from that of ALDH1a in the late embryo. At this stage, amphioxus ALDH1d expression is broad and inconspicuous with a moderate accentuation of the signal in the posterior gut endoderm. In C. intestinalis, ALDH1d expression is diffuse and weak in the gastrula and becomes diffusely transcribed in the trunk at 10–12 h of development. Thus, in both amphioxus and C. intestinalis, ALDH1a is consistently expressed in patterns that are entirely consistent with a role in anteroposterior patterning and that are reminiscent of expression of vertebrate ALDH1A2 (RALDH2), which defines posterior identity in the vertebrate embryo (31).

In both amphioxus and C. intestinalis, there is only a single gene encoding a narrow-channeled ALDH2 enzyme. In amphioxus, ALDH2 expression is restricted to posterior, mesendodermal tissues at 12 h of development and, subsequently, spreads throughout the embryo at 24 h (Fig. 2). In C. intestinalis, ALDH2 expression is strong and diffuse at early and late developmental stages (Fig. S4). Thus, ALDH2 genes are expressed in widespread patterns during development of invertebrate chordates.

There are a total of six genes encoding ALDH1s with narrow channels in amphioxus and C. intestinalis: amphioxus ALDH1b, ALDH1c, ALDH1e, and ALDH1f and C. intestinalis ALDH1b and ALDH1c. In amphioxus, these duplicates are weakly expressed in posterior domains overlapping that of ALDH1a in the neurula (at 12 h) (Fig. 2). However, by 24 h, they are expressed diffusely and weakly throughout the amphioxus embryo with a weak to moderate concentration of the signal for ALDH1b, ALDH1c, and ALDH1e in the posterior gut endoderm. In C. intestinalis, ALDH1b is diffusely transcribed in trunk and tail, whereas ALDH1c is not detectable by in situ hybridization in developing embryos (Fig. S4). Thus, the genes encoding narrow-channeled ALDH1s in amphioxus and C. intestinalis generally display either widespread or inconspicuous expression patterns during development, suggesting that these enzymes are not playing major roles in anteroposterior patterning of the embryo.