Hybridisation of Aldolase from <i>Drosophila</i>, Blocked at the Active Site, with Native C Aldolase from Calf Brain

Brenner‐Holzach, O.; Leuthardt, F.

doi:10.1111/j.1432-1033.1972.tb02548.x

Cited by 18 publications

(12 citation statements)

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“…Isolated hybrids of isozymes indicated that subunits are catalytically independent; for example, A3B1 was indistinguishable from an equivalent mixture of homotetramers, 3 A4: 1 B4 (14). Furthermore, hybrids of active and inactivated subunits also showed the independence of active sites (15).…”

mentioning

confidence: 96%

Disruption of the aldolase A tetramer into catalytically active monomers.

Beernink

Tolan

1996

Proc. Natl. Acad. Sci. U.S.A.

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The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Crosslinking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation.There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in. many proteins.

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mentioning

confidence: 96%

Disruption of the aldolase A tetramer into catalytically active monomers.

Beernink

Tolan

1996

Proc. Natl. Acad. Sci. U.S.A.

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“…Drosophila aldolase is 60 to 70% homologous to mammalian aldolases (27) and can functionally substitute for vertebrate subunits (4). The evidence presented here indicates that the Drosophila aldolase gene has organizational features in common with vertebrate genes.…”

Section: Discussionmentioning

confidence: 70%

Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster.

et al. 1992

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The fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster contains three divergent copies of an evolutionarily conserved 3' exon. Two mRNAs encoding aldolase contain three exons and differ only in the poly(A) site. The first exon is small and noncoding. The second encodes the first 332 amino acids, which form the catalytic domain, and is homologous to exons 2 through 8 of vertebrates. The third exon encodes the last 29 amino acids, thought to control substrate specificity, and is homologous to vertebrate exon 9. A third mRNA substitutes a different 3' exon (4a) for exon 3 and encodes a protein very similar to aldolase. A fourth mRNA begins at a different promoter and shares the second exon with the aldolase messages. However, two exons, 3a and 4a, together substitute for exon 3. Like exon 4a, exon 3a is homologous to terminal aldolase exons. The exon 3a-4a junction is such that exon 4a would be translated in a frame different from that which would produce a protein with similarity to aldolase. The putative proteins encoded by the third and fourth mRNAs are likely to be aldolases with altered substrate specificities, illustrating alternate use of duplicated and diverged exons as an evolutionary mechanism for adaptation of enzymatic activities.Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) is a glycolytic enzyme that catalyzes reversible aldol cleavage of FBP to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Aldolases from vertebrates, plants, microorganisms, and Drosophila spp. have been characterized (17). The enzymes from higher eukaryotes, including Drosophila spp., are type I aldolases, which function as tetramers and form a Schiffs base between the substrate and an active-site lysine residue. In vertebrates, three isozymes, A, B, and C, are expressed in different tissues. The Drosophila enzyme displays 60 to 70% sequence identity with the mammalian enzymes and is most related to the type A muscle forms (27). Drosophila enzyme subunits (39 kDa) can form functional heterotetramers with certain mammalian enzymes (4), indicating a high degree of functional conservation.Several mammalian aldolase genes (5,19,21,38,42,43) and a chicken aldolase gene (6) have been characterized, revealing close structural relationships between the genes encoding the different isozymes within a species and between the genes encoding equivalent isozymes in different species. The intron-exon organization is identical in all characterized vertebrate genes.In this report, we describe the organization of the aldolase gene of Drosophila melanogaster, which shares structural features with the vertebrate genes. The Drosophila gene appears unique in that it produces transcripts with alternate 3' ends, including one with two exons substituted for the terminal exon of the aldolase-encoding mRNAs. All terminal exons are related evolutionarily to the terminal exons of vertebrate aldolase-encoding mRNAs. The unique mRNAs, if translated, could produce aldolase-related proteins that * Corresponding author.contain th...

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“…Based on biochemical properties and primary sequence alignments it was suggested [4] that DALD is closest to the muscle type aldolases of vertebrates. DALD is able to form enzymatically active interspecies hybrids with vertebrate aldolases [5].…”

Section: Intrc Ductionmentioning

confidence: 99%

The crystal structure of fructose‐1,6‐bisphosphate aldolase fromDrosophila melanogaster at 2.5A˚resolution

Hester

Brenner‐Holzach

Rossi³

et al. 1991

FEBS Letters

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The structure of fructose-l,6-bisphosphate aldolase from Drosophila melanogaster has been determined by X-my diffraction at 2.5 .~ resolution.The insect enzyme crystallizes in space group P'2r2~2t with lattice constants a=86.60, b= 116.80, c= 151.58 ~. Molecular replacement with rabbit muscle aldolase as a search model has been employed to solve the structure. To improve the initial phases real space averaging, including phase extension from 4.0 to 2.5 ,/~, has been applied. Refinement of the atomic positions by molecular dynamics resulted in a crystallographic R-factor of 0.214. The tertiary structure resembles in most parts that of the vertebrate aldolase from rabbit muscle. Significant differences were found in surface loops and the N-and C-terminal regions of the protein. Here we present the first aldolase structure where the functionally important C-terminal arm is described completely.Crystal structure; Glycolytic enzyme; Fructose-1,6-bisphosphate aldolase; Substrate specificity; Drosophila melanogaster INTRC, DUCTIONFruetose-l,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) is a glycolytic enzyme catalyzing the reversible aldol cleavage of fructose-l,6-bisphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. FBP aldolase from Drosophila melanogaster (strain Sevelen, wild type, pupea) forms a Schiff base between the substrate and a lysyl residue [1] and belongs therefore to the class I aldolases [2]. Contrary to class I the class II aldolases use a divalent metal ion as cofactor [3]. The vertebrate aldolases (class I) comprise three isozymes. They refer to A (muscle), B (liver) and C (brain) depending on the tissue from which they are isolated [3]. The A and C form show a higher substrate specificity for FBP over fructose-l-phosphate (F1P), while the liver isozyme has no such preference.Abbreviations: DALD, fructose-l,6-bisphosphate aldolase from Drosophila melanogaster; RMALD, rabbit muscle aldolase; HMALD, human muscle aldolase; RLALD, rabbit liver aldolase; FBP, fructose-1,6-bisphosphate; FIP, fructose-l-phosphate; r.m.s., root-meansquare.Sequence numbering: Throughout this paper the sequence numbering of FBP aldolase from rabbit muscle will be applied.

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Hybridisation of Aldolase from Drosophila, Blocked at the Active Site, with Native C Aldolase from Calf Brain

Cited by 18 publications

References 20 publications

Disruption of the aldolase A tetramer into catalytically active monomers.

Disruption of the aldolase A tetramer into catalytically active monomers.

Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster.

The crystal structure of fructose‐1,6‐bisphosphate aldolase fromDrosophila melanogaster at 2.5A˚resolution

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Hybridisation of Aldolase from Drosophila, Blocked at the Active Site, with Native C Aldolase from Calf Brain

Cited by 18 publications

References 20 publications

Disruption of the aldolase A tetramer into catalytically active monomers.

Disruption of the aldolase A tetramer into catalytically active monomers.

Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster.

The crystal structure of fructose&#x2010;1,6&#x2010;bisphosphate aldolase fromDrosophila melanogaster at 2.5A˚resolution

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The crystal structure of fructose‐1,6‐bisphosphate aldolase fromDrosophila melanogaster at 2.5A˚resolution