Determination of some biochemical and structural features of alcohol dehydrogenases from Drosophila simulans and Drosophila virilis. Comparison of their properties with the Drosophila melanogaster Adhs enzyme.

The biochemical properties of the enzyme alcohol dehydrogenase of two different Drosophila species, Drosophila simulans and Drosophila virilis, were studied and compared with those of Drosophila melanogaster Adhs enzyme. All of them consist of two identical subunits of molecular weight 27800 and share significant similarities in function. The substrate specificities of these enzymes were characterized and Km(app.) and Vmax.(app.) values were calculated. All these alcohol dehydrogenases show greater affinity for secondary rather than for primary alcohols. The amino acid compositions of the three enzymes were determined, and there is a close similarity between the D. simulans and the D. melanogaster enzymes, but there are significant differences from the alcohol dehydrogenase of D. virilis. The N-terminal amino acid is blocked and the C-terminal amino acid is the same for all three alcohol dehydrogenases. The enzymes from the three species were carboxymethylated and digested with trypsin. The peptide 'maps' reveal, as expected, more homologies between the enzymes of D. simulans and D. melanogaster than with the enzyme of D. virilis.     

Alcohol dehydrogenase from Drosophila funebris and Drosophila immigrans: Molecular and evolutionary aspects

Alcohol dehydrogenase from Drosophila funebris and D. immigrans is evident at all developmental stages. The highest activity level appears in third-instar larvae and declines to a lower level at all later stages of development. Both species are monomorphic. The enzyme is a dimer consisting of two identical subunits with molecular weight 27,600. The pI values are 8.6 for D. funebris and 9.02 for D. immigrans. The optimum pH is 8.6 and 8.7 for D. funebris and D. immigrans, respectively. The Km values for NAD+, propan-2-ol, and butan-2-ol are 0.15, 2.90, and 2.08 mM, respectively, for D. funebris and 0.16, 1.53, and 1.49 mM, respectively, for D. immigrans. The half-life for the purified enzyme is 45 days for D. funebris and 18 days for D. immigrans at 4 degrees C. Data on the amino acid composition of both enzymes and peptide maps of alcohol dehydrogenase of D. immigrans reveal that they have marked homologies between them and also with alcohol dehydrogenases of other species. D. funebris shows reduced levels of alcohol dehydrogenase synthesis but has the highest specific activity reported to date for a Drosophila species. D. immigrans synthesises six times more enzyme but the specific activity is comparable to that of other species of Drosophila. This evidence could explain their different alcohol tolerance. The molecular properties of these alcohol dehydrogenases together with other species of Drosophila suggest that the alcohol dehydrogenase of Drosophila has arisen by divergent evolution from a common ancestral gene.     

Satellite Dnas in the Embryos of Various Species of the Genus Drosophila

A tentative evolutionary pattern has been found for two classes of the multiple satellite DNA's found in the genus Drosophila. The satellite DNA's from five Drosophila species (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) were analyzed and found to fall into three arbitrary CsCl buoyant density classes: Class I, rho = 1.661-1.669 g cm(-3), DNA molecules composed of primarily dA and dT moieties; Class II, rho = 1.685 and rho = 1.692, DNA molecules of low GC content; and Class III, rho = 1.711, a DNA of high GC composition. The dAT satellite DNA's appear in all the species studied except D. hydei, the species of most recent evolutionary divergence, whereas the heavy satellite appears only in the two species of most recent divergence, D. virilis and D. hydei.     

Kinetics and thermodynamics of ethanol oxidation catalyzed by genetic variants of the alcohol dehydrogenase from Drosophila melanogaster and D. simulans

Four naturally occurring variants of the alcohol dehydrogenase enzyme (ADH; EC 1.1.1.1) from Drosophila melanogaster and D. simulans, with different primary structures, have been subjected to kinetic studies of ethanol oxidation at five temperatures. Two amino acid replacements in the N-terminal region which distinguish the ADH of D. simulans from the three ADH allozymes of D. melanogaster generate a significantly different activation enthalpy and entropy, and Gibbs free energy change. The one or two amino acid replacements in the C-terminal region between the ADH allozymes of D. melanogaster do not have such clear-cut effects. All four ADH variants show highly negative activation entropies. Sarcosine oxidation by the ADH-71k variant of D. melanogaster has an activation energy barrier similar to that of ethanol oxidation. Three amino acid differences between the ADH of D. simulans and the ADH-F variant of D. melanogaster influence the kappa cat and kappa cat/Kethm constant by a maximum factor of about 2 and 2.5, respectively, over the whole temperature range. Product inhibition patterns suggest a 'rapid equilibrium random' mechanism of ethanol oxidation by the ADH-71k, and the ADH of D. simulans.     

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location.     

Interspecific analysis of the glycosidases of the sperm plasma membrane in Drosophila

We have studied the presence of four sperm glycosidases, alpha-mannosidase, alpha-L- fucosidase and two beta-hexosaminidase isoforms, in 11 species of the genus Drosophila spanning approximately an evolutionary 60 MY period, and in Scaptodrosophila lebanonensis, belonging to the ancestor genus Scaptodrosophila. These enzymes had been previously identified in Drosophila melanogaster as putative receptors for glycoconjugates of the egg surface. Alpha-mannosidase and beta-hexosaminidases are intrinsic proteins of the sperm plasma membrane in species closely related to D. melanogaster as well as in the divergent species D. willistoni, D. hydei, D. virilis, and S. lebanonensis. Alpha-L-fucosidase is restricted to the species of the genus Drosophila. Alpha-mannosidase and beta-hexosaminidases have been purified and characterized in all species. Their molecular masses and optimal pHs are similar in all the species, whereas interspecific differences in enzyme activities were detected. Cross-species comparison of kinetic parameters indicated a relationship between enzyme efficiency and phylogenetic relatedness. Beta-hexosaminidases were the most efficient enzymes. Lectin cytochemistry suggested the presence of carbohydrate residues complementary to the glycosidases on the eggshell at the site of sperm entry in all species. Bioinformatic analysis of the coding sequences of beta-hexosaminases and alpha-L-fucosidase and of their predicted products showed no evidence of positive selection of the genes coding for these enzymes and a high degree of sequence identity of the predicted polypeptides among the species of the genus Drosophila. Collectively, our findings indicate that the Drosophila sperm glycosidases are structurally and functionally conserved and strengthen the hypothesis of their involvement in the interactions with the egg surface.     

Molecular Population Genetics of an Electrophoretically Monomorphic Protein in the Alcohol Dehydrogenase Region of Drosophila Pseudoobscura

Nucleotide sequence data from the alcohol dehydrogenase (Adh) region of 18 isochromosomal strains of Drosophila pseudoobscura were used to determine whether the lack of amino acid polymorphism in ADH results from a low neutral mutation rate or a recent directional selection event. We estimated the neutral mutation parameter, 4Nmu, in synonymous sites for 17 subregions of Adh. The nucleotide diversity data were tested for departures from an equilibrium neutral model with two statistical tests. The Tajima test and the Hudson, Kreitman and Aguade test each failed to reject a neutral model. These results suggest that the ADH enzyme of D. pseudoobscura lacks amino acid polymorphisms because the neutral mutation rate of nonsynonymous sites is low. The neutral mutation parameter for synonymous sites is heterogeneous between domains of the Adh region. These data indicate that selective constrains on synonymous sites can vary between functional domains.     

Drosophila lebanonensis alcohol dehydrogenase: pH dependence of the kinetic coefficients

The alcohol dehydrogenase (ADH) from Drosophila lebanonensis shows 82% positional identity to the alcohol dehydrogenases from Drosophila melanogaster. These insect ADHs belong to the short-chain dehydrogenase/reductase family which lack metal ions in their active site. In this family, it appears that the function of zinc in medium chain dehydrogenases has been replaced by three amino acids, Ser138, Tyr151 and Lys155. The present work on D. lebanonensis ADH has been performed in order to obtain information about reaction mechanism, and possible differences in topology and electrostatic properties in the vicinity of the catalytic residues in ADHs from various species of Drosophila. Thus the pH dependence of various kinetic coefficients has been studied. Both in the oxidation of alcohols and in the reduction of aldehydes, the reaction mechanism of D. lebanonensis ADH in the pH 6-10 region was consistent with a compulsory ordered pathway, with the coenzymes as the outer substrates. Over the entire pH region, the rate limiting step for the oxidation of secondary alcohols such as propan-2-ol was the release of the coenzyme product from the enzyme-NADH complex. In the oxidation of ethanol at least two steps were rate limiting, the hydride transfer step and the dissociation of NADH from the binary enzyme-NADH product complex. In the reduction of acetaldehyde, the rate limiting step was the dissociation of NAD+ from the binary enzyme-NAD+ product complex. The pH dependences of the kon velocity curves for the two coenzymes were the opposite of each other, i.e. kon increased for NAD+ and decreased for NADH with increasing pH. The two curves appeared complex and the kon velocity for the two coenzymes seemed to be regulated by several groups in the free enzyme. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD+ complex, with a pKa value of 7.1. The kon velocity for acetaldehyde was pH independent and showed that in the enzyme-NADH complex, the pKa value of the catalytic residue must be above 10. The koff velocity of NAD+ appeared to be partly regulated by the catalytic residue, and protonation resulted in an increased dissociation rate. The koff velocity for NADH and the hydride transfer step was pH independent. In D. lebanonensis ADH, the pKa value of the catalytic residue was 0.5 pH units lower than in the ADHS alleloenzyme from D. melanogaster. Thus it can be concluded that while most of the topology of the active site is mainly conserved in these two distantly related enzymes, the microenvironment and electrostatic properties around the catalytic residues differ.     

Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase.

Drosophila alcohol dehydrogenase (ADH), an NAD(+)-dependent dehydrogenase, shares little sequence similarity with horse liver ADH. However, these two enzymes do have substantial similarity in their secondary structure at the NAD(+)-binding domain [Benyajati, C., Place, A. P., Powers, D. A. & Sofer, W. (1981) Proc. Natl Acad. Sci. USA 78, 2717-2721]. Asp38, a conserved residue between Drosophila and horse liver ADH, appears to interact with the hydroxyl groups of the ribose moiety in the AMP portion of NAD+. A secondary-structure comparison between the nucleotide-binding domain of NAD(+)-dependent enzymes and that of NADP(+)-dependent enzymes also suggests that Asp38 could play an important role in cofactor specificity. Mutating Asp38 of Drosophila ADH into Asn38 decreases Km(app)NADP 62-fold and increases kcat/Km(app)NADP 590-fold at pH 9.8, when compared with wild-type ADH. These results suggest that Asp38 is in the NAD(+)-binding domain and its substituent, Asn38, allows Drosophila ADH to use both NAD+ and NADP+ as its cofactor. The observations from the experiments of thermal denaturation and kinetic measurement with pH also confirm that the repulsion between the negative charges of Asp38 and 2'-phosphate of NADP+ is the major energy barrier for NADP+ to serve as a cofactor for Drosophila ADH.     

A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus.

An NAD+-dependent alcohol dehydrogenase (ADH) was purified to homogeneity from an aerobic strain of Bacillus stearothermophilus, DSM 2334 (ADH 2334), and compared with the ADH from B. stearothermophilus NCA 1503 (ADH 1503). When an antibody raised against ADH 2334 was used, no cross-reactivity with ADH 1503 was observed on Western blots; by means of an enzyme-linked-immunoabsorbent-assay ('e.l.i.s.a.') procedure, it was found that ADH 1503 had less than 6% of the antigenic activity of ADH 2334. Amino acid analyses detected very small differences in composition, equivalent to about 40 sequence changes, between the two enzymes. The new enzyme has the same six-amino-acid N-terminal sequence as ADH 1503. ADH 2334, but not ADH 1503, is reactive towards methanol; both enzymes can oxidize ethanol, propan-1-ol, butan-1-ol and butan-2-ol. The new enzyme has a distinctive pH optimum at pH 5.5-6 and has significantly lower KEthanolm and kEthanolcat. values than those of ADH 1503. From steady-state kinetic parameters of the reaction with ethanol, propan-1-ol and butan-1-ol, it was shown that ADH 2334 has an ordered mechanism in both directions, with NAD+ being the compulsory first substrate in alcohol oxidation and NADH release being the rate-limiting step. ADH 1503 has an ordered addition of NAD+ and alcohol, but NADH release is not rate-limiting.     

Isolation and characterization of a gene encoding DNA topoisomerase I in Drosophila melanogaster.

We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.     

DNA Sequence Comparison among Closely Related Drosophila Species in the MULLERI Complex

DNA hybridization was used to establish DNA sequence relationships among seven Drosophila species. Single-copy DNA was isolated from four species within the Drosophila mulleri complex, D. mojavensis, D. arizonensis, D. ritae and D. starmeri. These single-copy DNAs were used as tracers to be hybridized with each other and one additional member of the mulleri complex, D. aldrichi, a member of a closely related complex, D. hydei, and a distantly related species, D. melanogaster. Two methods have been used to determine the relatedness between these species: (1) the extent of duplex formed as measured by binding to hydroxyapatite and (2) the thermal stability of the duplexed DNA. Moderately repetitive DNA was purified from these species and used similarly to determine the divergence of this family of sequences. The rate of nucleotide substitution was estimated to be 0.2 +/-, 0.1% base pair change per million years for both single-copy and middle-repetitive DNAs. The size of the D. arizonensis genome, a representative of the mulleri complex, was calculated to be 2.2 X 10(8) base pairs from its kinetic complexity similar to that of D. hydei. The relative amounts (18%) and average reiteration frequency (100 copies) of the middle-repetitive DNA are similar for all Drosophila species studied. Finally, the data are presented in a phylogenetic tree.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Studies of esterase 6 in Drosophila melanogaster. XIII. Purification and characterization of the two major isozymes

Esterase-6 (EST 6; carboxylic-ester hydrolase; EC 3.1.1.1) from Drosophila melanogaster was purified to homogenity. Purified enzyme occurs as two closely moving isozymes, slow (EST 6S) and fast (EST 6F), on native polyacrylamide gel electrophoresis. Except for slight differences in their mobility, the two isozymes share similar molecular and catalytic properties. Both isozymes are glycoproteins and have an apparent molecular weight of 62,000 to 65,000 as judged by analytical gel filtration and sodium dodecyl sulfate (SDS) electrophoresis. They have identical mobility on SDS-polyacrylamide gels and an isoelectric point of 4.5. Each isozyme has a single active catalytic site as confirmed by titration with 0,0-diethyl-p-nitrophenyl phosphate (Paraoxon). We conclude that EST 6 is a monomeric enzyme. The amino acid composition of the two isozymes is very similar and both variants lack half-cystine residues. The low pI of the enzyme is due in part to a relatively high proportion of glutamic and aspartic amino acid residues. Characterization of the kinetic parameters of the isozymes using beta-naphthyl and p-nitrophenyl esters revealed no statistically significant differences in catalytic efficiency. There is, however, a suggestion that the two isozymes may differ in their substrate specificity.     

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.     

Circular DNA Molecules in the Genus Drosophila

The satellite DNA's from the embryos of five species of Drosophila (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) have been analyzed for the presence of closed circular duplex DNA molecules, as determined by CsCl-EBr gradients. Circular DNA molecules were found in every species but D. melanogaster. Analyses of cell fractions from adult Drosophila and organ fractions from Drosophila larvae show that fractions containing mitochondria are highly enriched in these molecules.     

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     

Regulation of the segmentation gene fushi tarazu has been functionally conserved in Drosophila.

An evolutionary approach was applied to identify elements involved in the regulation of the segmentation gene fushi tarazu (ftz) by comparing the Drosophila melanogaster ftz gene with its Drosophila hydei homologue. The overall organization of the ftz gene is very similar in both species. Surprisingly, ftz proved to be inverted in the ANT-C of D. hydei with respect to D. melanogaster. Strong homologies extend over the entire 6 kb of the ftz upstream region with the best match in the 'upstream element'. We identified several highly conserved boxes embedded in unrelated sequences that correspond extremely well to two germ layer specific enhancers in the upstream element. Transformation experiments revealed that D. hydei ftz gene products can restore D. melanogaster ftz function and, furthermore, that trans-acting factors from D. melanogaster recognize and control D. hydei ftz regulatory elements. These findings indicate a conservation of the entire regulatory network among segmentation genes for several millions of years during the evolution of Drosophila.     

Purification of mitochondrial NADH dehydrogenase from Drosophila hydei and comparison with the 'heat-shock' polypeptides.

Mitochondrial NADH dehydrogenase (NADH:(acceptor) oxidoreductase, EC .6.99.3) from either Drosophila hydei larvae or embryos has been purified 150- and 120-fold, respectively. The purified enzyme appeared homogeneous and showed a molecular weight of 57 000. The molecular weight of the nondenatured enzyme was 79 000. On isoelectro-focussing of the preparation, two fractions were observed, a major one with an isoelectric point of 6.2 and a minor fraction with an isoelectric point of 4.9. Straight-line kinetics in Lineweaver-Burk plots were observed for the purified enzyme with a Km of 0.040 mM. The Km was not changed during the purification procedure, suggesting that the enzyme was not denatured or inactivated. The pH optimum of the purified enzyme was 5.6. The molecular weight of the purified mitochondrial NADH dehydrogenase does not correspond to that of one of the 'heat-shock' polypeptides.