In order to investigate the class V ADH characteristics, we performed structural calculations. These methods, including MD simulations, have strength in giving insight into overall and local structural properties of proteins where, for different reasons, it is not possible to utilize traditional biochemical analyses. For human class I–IV ADH, and the corresponding structures from other mammals, all available crystal structures show a very high level of overall similarity. It is therefore not surprising that the generated models of human class III ADH, and class V ADH are very similar both when compared to their templates and when compared to each other. The TM-score of the comparison reference model (class III ADH) and its crystal structure (2fzw) was 0.76, implying that the modelling method is reliable for ADH structures.
However, after 20 ns of MD simulations, each chain of the class V ADH models formed an elongated β-strand in the dimerization region and the adjacent α-helix was destabilized in two out of three simulations (Fig. 1), the elongated β-strand matching pilot investigations done on the rat class V ADH . These structural elements are located in the region responsible for most of the subunit interactions (positions 290–320). It should be stressed that this was observed rarely in the structures of class I–IV ADH from any species. It was not observed in the reference models, and only in crystal structures of ADHs from single-cellular organisms and in mouse class II ADH. The latter is known to be inactive due to a mutation in another part of the enzyme, which could allow for further alterations . Elongated β-strands have also been observed in the tetrameric structure of yeast ADH , which has the 5-residue β-strand observed in class V ADH. However, the tetrameric formation is probably promoted by a 21-residue deletion in the region of the structural zinc which is not present in class V ADH, and thus probably not relevant to human class V ADH.
The formation of dimers has been shown to be essential for the stability of mammalian ADH enzymes , and a monomeric ADH enzyme has only been reported in one study . The elongated β-strands and the unfolded α-helices in this region imply that there are structural changes occurring at the dimerization region. The Ramachandran plots for the α-helices denote that Gly located at the start of the helices are forced into unfavoured conformations, which in turn implies that the simulations are attempting to make large changes to the structure.
The observed changes in the dimer formation region could be due to the alteration of a highly conserved Pro in class I–IV ADH to Gly in class V ADH. The Pro would be a clear divider between the β-strand and the α-helix, and could also support the formation of the α-helix, while the Gly would allow either type of secondary structure, elongating one and causing instability in the other. The unfolding of the α-helix could also be the result of the simulation compensating for structural bias caused by the homology modelling approach, where the initial model forced the protein into a slightly unfavoured conformation. The simulation could then be forced to compensate for the bias by forcing e.g. the Gly into an unfavoured conformation, explaining the Ramachandran outliers.
These observations, the elongation of the β-strands and the the instability of the α-helix, no matter if it is a real observation or caused by modelling bias, give a strong signal that the class V ADH structures have structural alterations at the dimer formation region. Even though this region has a varying set of residues between class I–IV, it has a very high structural conservation, and any alterations there could alter the stability of the rest of the structure [4, 6].
Class V ADH shows several differences in the conservation of certain amino acid residues when compared to other ADH enzymes (Table 1), which probably can explain some of the atypical behaviour of class V ADH.
Glu49, Met50, and Lys51 are located at the catalytic site. In class I and class IV ADH, this triplet is usually Asp-Asp-His, in class II the most common triplet is Asp-Ala-His, and in class III Asp-Ala-Tyr is almost perfectly conserved. Most sequences among class I–IV ADH have an aromatic residue (His/Tyr) at the third position (position 51 in Table 1), the only exception being a few class II ADH sequences with Thr instead. It has been shown that this position is involved both in the binding of the co-enzyme (NAD+) and the actual catalytic reaction . The size of Lys, along with the lack of an aromatic ring, makes this notable, as it could lead to a major alteration of the enzymatic activity or extinguish it as such. It should also be noted that this position is perfectly conserved within the class V ADH class, implying a specific function.
Cys265 (Cys in human class V, but Arg is the most common among the class V ADH enzymes; both are only present in class V ADH) is part of the central β-sheet. The position is class-unique with a high level of conservation within each class. The human residue, Cys, shares some properties with the residue types found in other classes (Ala/Ser/Thr), as opposed to the Arg that has a much higher level of conservation among the class V ADH proteins.
Leu295, Val299, and Gly305 are located in the region where most dimer interactions occur. Leu295 is located in the loop connecting the central β-sheet with the loop at the interaction site. The position is weakly conserved in class V ADH, where Ala and Pro are the most common residues among the other ADH classes. Val299 is one residue upchain of the elongated β-strand observed during the MD simulations. The residue is class-specific, with Gln, Lys, Glu, and Lys being the most common residue types in class I, class II, class III, and class IV, respectively. Gly305 is the class V-specific replacement of the fairly well-conserved Pro in class I–IV. It is located at the late turn of the loop in the dimer interaction region, one position from the short α-helix present in class I–IV. It may be involved in the structural alterations at the dimerization region.
The amino acid sequence of class V ADH shows ca. 60 % positional identity to those of the other ADHs, similar to that of the inter-class differences between any other ADH classes. For sequences analyzed in this study, the median and mean intra-class sequence identities among the class V ADH sequences are the lowest observed for any class; 70 and 75 % (Table 2), but the levels are in the vicinity of those of other classes, e.g. class II ADH: 74 and 77 %, respectively. While there is a variation among the species represented within each class, there is a slightly lower intra-class similarity between the class V ADH sequences than among the other classes.
This tendency could also be observed when calculating the evolutionary pressure, dN/dS , on the different ADH enzymes. class III had the strongest evolutionary pressure with a dN/dS ratio of 0.126, followed by class IV, class I, class II, and finally class V at 0.385, placing the class V ADH dN/dS ratio higher than that of many other proteins, but far below the level of some highly variably proteins with both specific and important functions.
Class III ADH is known to be the ancestral form of the zinc-containing ADHs  and shows a crucial function in glutathione dependent formaldehyde scavenging as well as in NO metabolism. The enzyme has been detected, so far, in all species harbouring glutathione from bacteria to primates , and as such, a high evolutionary pressure was expected.
The other well-characterized ADHs are involved in general alcohol dehydrogenase activities, where the class I ADH enzymes tend to have a general detoxifying function, aiding in the metabolism of many alcohols including ethanol and hydroxysteroids. Class II ADH is believed to be involved in the metabolism of retinols and aromatic alcohols, e.g. hydroquinone. Worth noting is that some rodent species have a Pro at position 47, rendering their enzymatic activity near non-existent . Finally, class IV is the main ADH involved in the retinoid metabolism, e.g. in cell differentiation , and in addition in the first-pass ethanol metabolism in humans .
Class V ADH can be expressed under certain conditions, as shown from Northern blot, in vitro translation and proteomics analyses [11, 12]. However, a stable and active protein has never been reported. Fused recombinant class V ADH has been expressed both as GFP-class V ADH and GST-class V ADH. In none of these cases any activity with known substrate for ADHs was traced , where other mammalian ADHs under similar conditions showed activity. For other protein families, pseudoenzymes have been ascribed as integral parts of the cell system, where they today play unknown roles .
Based on the functions of the enzymes, it can be concluded that the dN/dS ratios make sense, the enzymatic specificity having a correlation to the level of evolutionary pressure . As e.g. class II ADH has a lower dN/dS ratio than class V ADH, even with some species having a common variant with near no activity, it implies that some (or all) species may lack activity in class V ADH as well. However, as the calculated value for class V ADH was still much lower than 1 (0.385), it indicates that there is an evolutionary pressure on the class V ADH sequence with a bias towards eliminating non-functional mutations, and class V ADH thereby has a supposed, but currently unknown, function in humans.
The phylogenetic tree shows that class V and class VI ADH are unique and separate classes that should not be mixed up, confirming earlier results .