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

Juan, Elvira; Gonzàlez‐Duarte, Roser

doi:10.1042/bj1950061

Cited by 22 publications

(10 citation statements)

References 18 publications

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“…Phylogenetic relationships based on the amino acid composition also suggest that D. lebanonensis is not closely related to D. melanogaster (Vilageliu & Gonzalez-Duarte, 1984). Table 3 shows that the substrate stereospecificity of D. lebanonensis Adh is similar to that of other Drosophila species Adh (Chambers et al, 1981;Juan & Gonzalez-Duarte, 1981;Oakeshott et al, 1982;Winberg et al, 1982a;Hovik et al, 1984), with secondary alcohols being better substrates than primary alcohols. This implies that all Drosophila alcohol dehydrogenases studied so far contain a substrate-binding region where both alkyl groups of a secondary alcohol are bound to the enzyme.…”

Section: Discussionmentioning

confidence: 74%

“…The amino acid composition of the protein was determined according to Juan & Gonzalez-Duarte (1981). Protein hydrolysates were analysed on a Chromaspek J 180 Rank Hilger automatic amino acid analyser equipped with a fluorimeter.…”

Section: Amino Acid Analysismentioning

confidence: 99%

“…Drosophila alcohol dehydrogenases contain two subunits, each with an Mr of about 27000 (Schwartz et al, 1975;Thatcher, 1980;Juan & Gonzalez-Duarte, 1981), and have higher activity with secondary than with primary alcohols (Vigue & Johnson, 1973;Daggard, 1981;Juan & Gonzalez-Duarte, 1981;Oakeshott et al, 1982).…”

Section: Introductionmentioning

confidence: 99%

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Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis

Winberg¹,

Hovik²,

McKinley-McKee³

et al. 1986

Biochemical Journal

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Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.

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Section: Discussionmentioning

confidence: 74%

Section: Amino Acid Analysismentioning

confidence: 99%

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Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis

Winberg¹,

Hovik²,

McKinley-McKee³

et al. 1986

Biochemical Journal

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“…In accordance with the need for an alkaline lateral chain in position 156, to stabilize the coenzyme or/and lower the pK a of the Y152 hydroxyl group, the mutant K156I has been shown to be inactive (Cols et al 1993;Chen et al 1993), while K156R retained 2.2% of its activity. Surprisingly, a glutamic acid at position 156 of D. virilis ADH has been reported (Nurminsky et al 1996), although this enzyme exhibits a reaction behavior similar to that of other Drosophila species (Juan and Gonzàlez-Duarte 1980). Recently we have also shown the null activity of the S139A and S139C enzymes (Cols et al 1997).…”

Section: Discussionmentioning

confidence: 80%

Shaping of Drosophila Alcohol Dehydrogenase Through Evolution: Relationship with Enzyme Functionality

et al. 1998

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Drosophilidae is a large, widely distributed family of Diptera including 61 genera, of which Drosophila is the most representative. Drosophila feeding is part of the saprophytic trophic chain, because of its dependence upon decomposing organic matter. Many species have adapted to fermenting fruit feeding or to artificial (man-made) fermentation habitats, such as cellars and breweries. Actually, the efficient exploitation of niches with alcohols is considered one of the reasons for the worldwide success of this genus. Drosophila alcohol dehydrogenase (ADH), a member of the short-chain dehydrogenase/reductase family (SDR), is responsible for the oxidation of alcohols, but its direct involvement in fitness, including alcohol tolerance and utilization, gives rise to much controversy. Thus, it remains unclear whether ADH differentiation through evolution is somehow associated with natural adaptation to new feeding niches, and thus maybe to Drosophila speciation, or if it is a simple reflection of neutral divergence correlated with time separation between species. To build a hypothesis which could shed light on this dilemma, we analyzed the amino acid variability found in the 57 protein ADH sequences reported up to now, identified the taxon-specific residues, and localized them in a three-dimensional ADH model. Our results define three regions whose shaping has been crucial for ADH differentiation and would be compatible with a contribution of ADH to Drosophila speciation.

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“…Thus, 50 % of activity was retained at 0.6 M-GdnHCI; in the absence of NAD+ only 10% of activity was retained. The concentration of NAD+ used in all the protection experiments was 220 AM, a value corresponding to the Km of NAD+ for Drosophila ADH (Juan & Gonzalez-Duarte, 1981).…”

Section: Unfolding Of Drosophila Adh By Gdnhcimentioning

confidence: 99%

Structural properties of long- and short-chain alcohol dehydrogenases. Contribution of NAD+ to stability

Pouplana

Atrian

Gonzàlex-Duarte

et al. 1991

Biochemical Journal

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Structural studies were undertaken on long-chain and short-chain alcohol dehydrogenases (from horse liver and Drosophila respectively). Far-u.v. c.d. measurements were used to estimate the secondary structure contents of the enzymes. For the horse liver enzyme, the results agree well with the X-ray data; for the Drosophila enzyme (for which a crystal structure is not yet available), the results are in good agreement with those obtained by applying a range of structure-prediction procedures to the amino acid sequence of this enzyme. The conformational stabilities of the two enzymes were investigated by studying the unfolding brought about by guanidinium chloride (GdnHCl) by using activity and c.d. measurements. The unfolding of the Drosophila enzyme was analysed in terms of a two-state model; the presence of the substrate NAD+ leads to considerable protection against unfolding. By contrast, the unfolding of the horse liver enzyme shows a plateau effect at intermediate concentrations of GdnHCI, indicating that a two-state model is not appropriate in this case. NAD+ affords little, if any, protection against unfolding for the horse liver enzyme.

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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

Cited by 22 publications

References 18 publications

Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis

Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis

Shaping of Drosophila Alcohol Dehydrogenase Through Evolution: Relationship with Enzyme Functionality

Structural properties of long- and short-chain alcohol dehydrogenases. Contribution of NAD+ to stability

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