ADH and phylogenetic relationships ofDrosophila lebanonensis (Scaptodrosophila)

Summary Increasing data onDrosophila alcohol dehydrogenase (ADH) sequences have made it possible to calculate the rate of amino acid replacement per year, which is 1.7×10^–9. This value makes this protein suitable for reconstructing phylogenetic relationships within the genus for those species for which no molecular data are available such asScaptodrosophila. The amino acid sequence ofDrosophila lebanonensis is compared to all of the already knownDrosophila ADHs, stressing the unique characteristic features of this protein such as the conservation of an initiating methionine at the N-terminus, the unique replacement of a glycine by an alanine at a very conserved position in the NAD domain of all dehydrogenases, the lack of a slowmigrating peptide, and the total conservation of the maximally hydrophilic peptide. The functional significance of these features is discussed.Although the percent amino acid identity of the ADH molecule inDrosophila decreases as the number of sequences compared increases, the conservation of residue type in terms of size and hydrophobocity for the ADH molecule is shown to be very high throughout the genusDrosophila. The distance matrix and parsimony methods used to establish the phylogenetic relationships ofD. lebanonensis show that the three subgenera,Scaptodrosophila, Drosophila, andSophophora separated at approximately the same time.     

Drosophila fat body protein P6 and alcohol dehydrogenase are derived from a common ancestral protein

Summary Drosophila melanogaster alcohol dehydrogenase is an example of convergent evolution: it is not related to the ADHs of other organisms, but to short-chain dehydrogenases, which until now have been found only in bacteria and in mammalian steroid hormone metabolism. We present evidence that theDrosophila ADH is phylogenetically more closely related to P6, another highly expressed protein from the fat body ofDrosophila, than it is to the short-chain dehydrogenases. The polypeptide sequence of P6 was inferred from DNA sequence analysis. Both ADH and P6 polypeptides have retained a high structural similarity with respect to the Chou-Fasman prediction of secondary structure and hydropathy. P6 is also homologous to the 25-kd protein from the fat body ofSarcophaga peregrina, whose sequence we have reexamined. The evolution of the P6-ADH family of proteins is characterized by a dramatic increase in the methionine content of P6. Methionine accounts for 20% of P6 amino acids. This is in contrast with the absence of this amino acid in mature ADH. There is evidence that P6 and the 25-kd protein have undergone a parallel and independent enrichment in methionine. When corrected for this, the rate of amino acid replacement shows that the P6-25-kd lineage diverged from insect ADH shortly before the divergence of the ADH gene (Adh) from its 3-duplication (Adhdup).     

Molecular evolution of mammalian class I alcohol dehydrogenase

Phylogenetic relationship and the rates of evolution of mammalian alcohol dehydrogenases (ADHs) have been studied by using the amino acid sequences from the human (ADH alpha, ADH beta, and ADH gamma), rat, mouse, and horse (ADH E and ADH S). With the maize ADH1 and ADH2 used as references, the patterns of the amino acid replacements in the beta-sheets, alpha-helices, and random coils in each of the catalytic and coenzyme-binding domains were analyzed separately. The phylogenetic trees based on the different sets of amino acid substitutions consistently showed that (1) multiple ADHs in human and horse have arisen after mammalian radiation, (2) the common ancestor of human ADHs alpha and beta diverged from the ancestor of ADH gamma first and the former two ADHs diverged from each other more recently, and (3) the human ADHs are more closely related to the rodent ADHs than to the horse ADHs. Furthermore, the estimated branch lengths showed that the rodent ADHs are evolving faster than the other ADHs. This difference in evolutionary rate between the two groups of organisms is explainable either in terms of the difference in the number of cell generations per year or in terms of reduction of functional constraints.     

Selection and Methionine Accumulation in the Fat Body Protein 2 Gene (FBP2), a Duplicate of the Drosophila Alcohol Dehydrogenase (ADH) Gene

The Drosophila fat body protein 2 gene (Fbp2) is an ancient duplication of the alcohol dehydrogenase gene (Adh) which encodes a protein that differs substantially from ADH in its methionine content. In D. melanogaster, there is one methionine in ADH, while there are 51 (20% of all amino acids) in FBP2. Methionine is involved in 46% of amino acid replacements when Fbp2 DNA sequences are compared between D. melanogaster and D. pseudoobscura. Methionine accumulation does not affect conserved residues of the ADH-ADH^r-FBP2 multigene family. The multigene family has evolved by replacement of mildly hydrophobic amino acids by methionine with no apparent reversion. Its short-term evolution was compared between two Drosophila species, while its long-term evolution was compared between two genera belonging respectively to acalyptrate and calyptrate Diptera, Drosophila and Sarcophaga. The pattern of nucleotide substitution was consistent with an independent accumulation of methionines at the Fbp2 locus in each lineage. Under a steady-state model, the rate of methionine accumulation was constant in the lineage leading to Drosophila, and was twice as fast as that in the calyptrate lineage. Substitution rates were consistent with a slight positive selective advantage for each methionine change in about one-half of amino acid sites in Drosophila. This shows that selection can potentially account for a large proportion of amino acid replacements in the molecular evolution of proteins. Received: 12 December 1994 / Accepted: 15 April 1996     

Origin and Evolution of a New Gene Descended from Alcohol Dehydrogenase in Drosophila

Drosophila alcohol dehydrogenase (Adh) is highly conserved in size, organization, and amino acid sequence. Adh-ψ was hypothesized to be a pseudogene derived from an Adh duplication in the repleta group of Drosophila; however, several results from molecular analyses of this gene conflict with currently held notions of molecular evolution. Perhaps the most difficult observations to reconcile with the pseudogene hypothesis are that the hypothetical replacement sites of Adh-ψ evolve only slightly more quickly than replacement sites of closely related, functional Adh genes, and that the replacement sites of the pseudogenes evolve considerably more slowly than neighboring silent sites. The data have been presented as a paradox that challenges our understanding of the mechanisms underlying DNA sequence divergence. Here I show that Adh-ψ is actually a new, functional gene recently descended from an Adh duplication. This descendant recruited ~60 new N-terminal amino acids, is considerably more basic than ADH, and is evolving at a faster rate than Adh. Furthermore, though the descendant is clearly functional, as inferred from molecular evolution and population genetic data, it retains no obvious ADH activity. This probably reflects functional divergence from its Adh ancestor.     

Similarities in protein amino acid composition in connection with principles of protein evolution

The amino acid composition of human alcohol dehydrogenase (ADH) was compared with alcohol dehydrogenases from different organisms and with other proteins. Similar amino acid sequences in human ADH (template protein) and in other proteins were determined by means of an original computer program. Analysis of amino acid motifs reveals that the ADHs from evolutionary more close organisms have more common amino acid sequences. The quantity measure of amino acid similarity was the number of similar motifs in analyzed protein per protein length. This value was measured for ADHs and for different proteins. For ADHs, this quotient was higher than for proteins with different functions; for vertebrates it correlated with evolutionary closeness. The similar operation of motif comparison was made with the help of program complex “MEME”. The analysis of ADHs revealed 4 motifs common to 6 of 10 tested organisms and no such motifs for proteins of different function. The conclusion is that general amino composition is more important for protein function than amino acid order and for enzymes of similar function it better correlates with evolutionary distance between organisms.     

Protein evolution in different cellular environments: cytochrome b in sharks and mammals

DNA sequences for the mitochondrial cytochrome b gene were determined for 13 species of sharks. Rates and patterns of amino acid replacement are compared for sharks and mammals. Absolute rates of cytochrome b evolution are six times slower in sharks than in mammals. Bivariate plots of the number of nonsynonymous and silent transversions are indistinguishable in the two groups, however, suggesting that the differences in amino acid replacement rates are due primarily to differences in DNA substitution rates. Patterns of amino acid replacement are also similar in the two groups. Conserved and variable regions occur in the same parts of the cytochrome b gene, and there is little evidence that the types of amino acid changes are significantly different between the groups. Similarity in the relative rates and patterns of protein change between the two groups prevails despite dramatic differences in the cellular environments of sharks and mammals. Poor penetrance of physiological differences through to rates of protein evolution provides support for the neutral theory and suggests that, for cytochrome b, patterns of evolution have been relatively constant throughout much of vertebrate history.      

An empirical codon model for protein sequence evolution

In the past, 2 kinds of Markov models have been considered to describe protein sequence evolution. Codon-level models have been mechanistic with a small number of parameters designed to take into account features, such as transition-transversion bias, codon frequency bias, and synonymous-nonsynonymous amino acid substitution bias. Amino acid models have been empirical, attempting to summarize the replacement patterns observed in large quantities of data and not explicitly considering the distinct factors that shape protein evolution. We have estimated the first empirical codon model (ECM). Previous codon models assume that protein evolution proceeds only by successive single nucleotide substitutions, but our results indicate that model accuracy is significantly improved by incorporating instantaneous doublet and triplet changes. We also find that the affiliations between codons, the amino acid each encodes and the physicochemical properties of the amino acids are main factors driving the process of codon evolution. Neither multiple nucleotide changes nor the strong influence of the genetic code nor amino acids' physicochemical properties form a part of standard mechanistic models and their views of how codon evolution proceeds. We have implemented the ECM for likelihood-based phylogenetic analysis, and an assessment of its ability to describe protein evolution shows that it consistently outperforms comparable mechanistic codon models. We point out the biological interpretation of our ECM and possible consequences for studies of selection.     

Gnarled-trunk evolutionary model of influenza A virus hemagglutinin

Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.     

Functional Constraints of Alcohol Dehydrogenase (ADH) of Tephritidae and Relationships with Other Dipteran Species

Alcohol dehydrogenase is considered a very important enzyme in insect metabolism because it is involved (in its homodimeric form) in the catalysis of the reversible conversion of various alcohols in larval feeding sites to their corresponding aldehydes and ketones, thus contributing to detoxification and metabolic purposes. Using 14 amino acid ADH sequences recently determined in our laboratory, we constructed a three-dimensional (3D) model of olive fruit fly Bactrocera oleae ADH1 and ADH2, based on the known homologous Drosophila lebanonensis ADH structure, and the amino acid residues that have been proposed as being responsible for catalysis were located on it. Moreover, in a comparative study of the ADH sequences, the residues occupying characteristic positions in the ADH of species of the Bactrocera and Ceratitis genera (called genus-specific) as well as residues appearing only in ADH1 or ADH2 (called isozymic-specific) were defined and localized on the 3D model. All regions important for catalytic activity, such as those forming the substrate- and coenzyme-binding sites, are highly conserved in all tephritid species examined. Genus-specific amino acids are located on the outside of the protein, on loops and regions predicted to be antigenic. The higher percentage of genus-specific amino acid variation seems to be centered in the NAD adenine-binding site, located near the surface of the protein molecule. Nine of 12 isozymic-specific positions are lined along an arc on the surface of the protein, thus linking the two monomer bases of the dimer via the C-terminal interacting loops. Furthermore, the distribution of isozymic- and genus-specific amino acids on the monomer–monomer interface may have some evolutionary significance. Most amino acids predicted to be antigenic are positioned in peripheral regions of nonfunctional importance, but surprisingly, an additional antigenic region is contained within the (highly conserved in tephritids) C-terminal tail.     

Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development

In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity.     

Protein evolution within and between species

Protein evolution can be seen as the successive replacement of amino acids by other amino acids. In general, it is a very slow process which is triggered by point mutations in the nucleotide sequence. These mutations can transform into single nucleotide polymorphisms (SNPs) within populations and diverging proteins between species. It is well known that in many cases amino acids can be replaced by others without impeding the functioning of the protein, even if these are of quite different physico-chemical character. In some cases, however, almost any replacement would result in a functionally deficient protein. Based upon comprehensive published SNP data and applying correlation analysis we quantified the two antagonist factors controlling the process of amino acid replacement and thus protein evolution: First, the degenerate structure of the genetic code which facilitates the exchange of certain amino acids and, second, the physico-chemical forces which limit the range of possible exchanges to maintain a functional protein. We found that the observed frequencies of amino acid exchanges within species are best explained by the genetic code and that the conservation of physico-chemical properties plays a subordinate role, but has nevertheless to be considered as a key factor. Between moderately diverged species genetic code and physico-chemical properties exert comparable influence on amino acid exchanges. We furthermore studied amino acid exchanges in more detail for six species (four mammals, one bird, and one insect) and found that the profiles are highly correlated across all examined species despite their large evolutionary divergence of up to 800 million years. The species specific exchange profiles are also correlated to the exchange profile observed between different species. The currently available huge body of SNP data allows to characterize the role of two major shaping forces of protein evolution more quantitatively than before.     

Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5

BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.     

Testing for Spatial Clustering of Amino Acid Replacements Within Protein Tertiary Structure

Widely used models of protein evolution ignore protein structure. Therefore, these models do not predict spatial clustering of amino acid replacements with respect to tertiary structure. One formal and biologically implausible possibility is that there is no tendency for amino acid replacements to be spatially clustered during evolution. An alternative to this is that amino acid replacements are spatially clustered and this spatial clustering can be fully explained by a tendency for similar rates of amino acid replacement at sites that are nearby in protein tertiary structure. A third possibility is that the amount of clustering exceeds that which can be explained solely on the basis of independently evolving protein sites with spatially clustered replacement rates. We introduce two simple and not very parametric hypothesis tests that help distinguish these three possibilities. We then apply these tests to 273 homologous protein families. The null hypothesis of no spatial clustering is rejected for 102 of 273 families. The explanation of spatially clustered rates but independent change among sites is rejected for 43 families. These findings need to be reconciled with the common practice of basing evolutionary inferences on models that assume independent change among sites. [Reviewing Editior: Dr. David Pollock]     

On the PAM matrix model of protein evolution

The internal consistency of the PAM matrix model of protein evolution is here investigated. The 1 PAM matrix has been constructed from amino acid replacements observed in closely related sequences. Such replacements are of two types, those that do not require an intermediate amino acid replacement and those that do. The second type of replacement must generally be produced by a repetition of the first. This allows data on the first type to be used in predicting data on the second type so that some elements of the 1 PAM matrix may be used to predict others. A discrepancy of more than two orders of magnitude is found between the predictions and the data when this is carried out. This is partly accounted for by an error in constructing the matrix. However, it also seems necessary that the basic model be modified. Several possibilities are considered. One of these is to incorporate a site-dependent spectrum of mutabilities associated with each amino acid.      

Sequence analysis of two null-mutant alleles of the single Arabidopsis Adh locus

Summary Data presented in this paper deal with a further molecular characterization of 2 out of 32 EMS-induced Arabidopsis ADH null mutants that we isolated previously. In order to localize and characterize each mutation at the molecular level, we have cloned and completely sequenced the R002 and R006 null mutant alleles. For mutant R002, which does not contain any detectable levels of ADH protein and mRNA, we have found that the mutation is due to a single C to T base pair substitution in the reading frame; this leads to the incorporation of a TAG stop codon (amber nonsense mutation). For mutant R006, which contains normal levels of inactive protein and mRNA levels, we found a G to A base pair transition. This gives rise to a Cys to Tyr amino acid substitution in the active site of the ADH enzyme.Abbreviations CRM cross-reacting material - 2,4-D 2,4-dichlorophenoxyacetic acid - EMS ethylmethanesulfonate     

Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart.

Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M. (1992) J. Biol. Chem. 267, 10184-10192). The identity in the amino acid sequences of these enzymes is 52%. As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability. A variety of mutant proteins of E. coli RNase HI were constructed and analyzed for protein stability. In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T. thermophilus RNase H. Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability. Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative. Replacement of all four regions (R4-R7) gave the most stable mutant protein. The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E. coli RNase HI. These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner.     

Recent origin for a thermostable alcohol dehydrogenase allele ofDrosophila melanogaster

Summary The nucleotide sequence of theFast-Chateau Douglas isolate of the thermostable alcohol dehydrogenase allele is compared with the sequences of theSlow andFast alleles ofDrosophila melanogaster. Conceptual translation of theFChD sequence indicates that the thermostable polypeptide has the diagnostic FAST amino acid replacement at residue 192 and an additional replacement of serine for proline at residue 214. This suggests aFast origin for the thermostableAdh allele. However, some of the biochemical properties of the FCHD protein resemble those of the SLOW rather than the FAST polypeptides. The serine for proline replacement confers upon the thermostable polypeptide substrate specificities and some kinetic parameters similar to the SLOW protein. The same replacement substitution within the third coding exon also appears to alter the ADH protein concentration to a level similar to the SLOW polypeptide and the probable effect is at the level of mRNA concentration. The low level of nucleotide sequence variation, other than that leading to the amino acid substitution, suggests a recent origin for the thermostable allele. The time since divergence of theFChD sequence fromFast is estimated to be approximately 260,000–470,000 years.