The predicted secondary structure of enolase.

The results of several secondary-structure prediction programs were combined to produce an estimate of the regions of alpha-helix, beta-sheet and reverse turn for both chicken skeletal-muscle and yeast enolase sequences. The predicted secondary-structure content of the chicken enzyme is 27% alpha-helix and less than 10% beta-sheet, whereas in the yeast enolase a similar helix content but virtually no sheet are predicted. These results are in fair agreement with published experimental estimates of the amount of secondary structure in the yeast enzyme. The enzyme appears to be formed from three domains.     

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.     

Fructose-1,6-bisphosphate aldolase from Drosophila melanogaster: primary structure analysis, secondary structure prediction, and comparison with vertebrate aldolases

The amino acid sequence of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster was determined and was compared with those of five vertebrate aldolases on record. The four identical polypeptide chains of the insect enzyme, acetylated at the N-terminus and three residues shorter than the vertebrate chains, contain 360 amino acid residues. Of these 190 (or 53%) are identical in all six enzymes and in addition 33 positions (or 9%) are occupied by homologous residues. Comparison with the muscle-type isoaldolases from man and rabbit and the liver-type isoaldolases from man, rat, and chicken indicates an average sequence identity of 70 and 63%, respectively. Thus, the insect and the vertebrate muscle aldolases are probably coded by orthologous genes. On this basis an average rate of evolution of 3.0 PAM per 10(8) years is calculated, documenting an evolutional divergence slower than that of cytochrome c (4.2 PAM/10(8) years). The rate is also lower than that of the liver isoform (3.6 PAM/10(8) years). Secondary structure prediction analysis for Drosophila aldolase suggests the occurrence of 11-12 helical segments and 8-9 beta-strands. The conspicuous alternation of these structures in all six aldolases, especially in the C-terminal 200 residues, is consistant with the formation of an alpha beta-barrel supersecondary structure as documented for several other glycolytic enzymes.     

Prediction of secondary structure by evolutionary comparison: application to the alpha subunit of tryptophan synthase

The amino acid sequences of the a subunits of tryptophan synthase from ten different microorganisms were aligned by standard procedures. The alpha helices, beta strands and turns of each sequence were predicted separately by two standard prediction algorithms and averaged at homologous sequence positions. Additional evidence for conserved secondary structure was derived from profiles of average hydropathy and chain flexibility values, leading to a joint prediction. There is good agreement between (1) predicted beta strands, maximal hydropathy and minimal flexibility, and (2) predicted loops, great chain flexibility, and protein segments that accept insertions of various lengths in individual sequences. The a subunit is predicted to have eight repeated beta-loop-alpha-loop motifs with an extra N-terminal alpha helix and an intercalated segment of highly conserved residues. This pattern suggests that the territory structure of the a subunit is an eightfold alpha/beta barrel. The distribution of conserved amino acid residues and published data on limited proteolysis, chemical modification, and mutagenesis are consistent with the alpha/beta barrel structure. Both the active site of the a subunit and the combining site for the beta 2 subunit are at the end of the barrel formed by the carboxyl-termini of the beta strands.     

The crystal structure of human muscle aldolase at 3.0 A resolution

The three-dimensional structure of fructose-1,6-bisphosphate aldolase from human muscle has been determined at 3.0 A resolution by X-ray crystallography. The active protein is a tetramer of 4 identical subunits each of which is composed of an eight-stranded alpha/beta-barrel structure. The lysine residue responsible for Schiff base formation with the substrate is located near the centre of the barrel in the middle of the sixth beta-strand. While the overall topology of the alpha/beta-barrel is very similar to those found in several other enzymes, the distribution of charged residues inside the core of the barrel seems distinct. The quaternary fold of human muscle aldolase uses interfacial regions also involved in the subunit association of other alpha/beta-barrel proteins found in glycolysis, but exploits these regions in a manner not seen previously.     

The crystal structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster at 2.5 A resolution

The structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster has been determined by X-ray diffraction at 2.5 A resolution. The insect enzyme crystallizes in space group P2(1)2(1)2(1) with lattice 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 A, 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.     

Structure, calmodulin-binding, and calcium-binding properties of recombinant alpha spectrin polypeptides

We examined the structure and the distribution of binding activities within bacterially produced fragments of Drosophila alpha spectrin. By electron microscopy, purified spectrin fragments resembled the corresponding regions of native spectrin. The contour lengths of recombinant spectrin molecules were proportional to the length of their coding sequences, which is consistent with current models of spectrin structure in which individual segments of the polypeptide contribute independently to the structure of the native molecule. We localized two sites at which calcium may regulate spectrin function. First, a site responsible for calmodulin binding to Drosophila alpha spectrin was identified near the junction of repetitive segments 14 and 15. Second, a domain of Drosophila alpha spectrin that includes two EF hand calcium-binding sequences bound 45Ca in blot overlay assays. EF hand sequences from a homologous domain of Drosophila alpha actinin did not bind calcium under the same conditions.     

Identification of a molecular target for the calcium-modulated protein S100. Fructose-1,6-bisphosphate aldolase

A rat brain S100-binding protein, R40,000, has been isolated, characterized, and identified as fructose-1,6-bisphosphate aldolase. R40,000 was purified by ammonium sulfate precipitation, hydroxylapatite chromatography, dye-binding chromatography, and electroelution from sodium dodecyl sulfate-polyacrylamide gels. Microsequence analysis of a fragment of R40,000 revealed a 15-residue amino acid sequence which shows a high degree of homology to the amino acid sequence of fructose-1,6-bisphosphate aldolase from rabbit muscle and rat liver. Further characterization demonstrated that R40,000 has an amino acid composition, subunit molecular weight, and cyanogen bromide map similar to aldolase. In addition, purified aldolase interacts with S100 alpha and S100 beta by gel overlay, and aldolase enzyme activity is stimulated 2-fold in vitro by S100 alpha and S100 beta. S100 interacts predominantly with the C or brain-specific form of the enzyme in gels and stimulates the activity of the C-enriched form of the enzyme in a calcium-dependent manner. Altogether, these data suggest that fructose-1,6-bisphosphate aldolase may be an intracellular target of S100 action in brain.     

Quaternary structure of aldolase leads to differences in its folding and unfolding intermediates

Pulsed hydrogen exchange mass spectrometry has been used to investigate folding of rabbit muscle aldolase, an alpha/beta-barrel protein exhibiting the classic TIM structure. Aldolase unfolded in GdHCl refolded as the denaturant concentration was reduced by dialysis. Samples withdrawn during dialysis were pulse-labeled with deuterium to identify unfolded regions in structural forms highly populated during the folding process. Intact, labeled aldolase was digested into fragments, which were analyzed by HPLC electrospray ionization mass spectrometry to detect the H/D exchange along the aldolase backbone. For some concentrations of GdHCl, bimodal distributions of deuterium were found for most peptic fragments, indicating that regions represented by these fragments were either unfolded or folded in the intact polypeptide prior to labeling. The extent of folding was determined from these mass spectra, as well as by CD (220 nm) and enzymatic activity. These results show that folding to the active form involves three domains and two intermediates. Approximately 110 residues fold to highly compact forms in each step. These results also show that each folding domain includes widely separated regions of the backbone. When compared with the results of a previous study of aldolase unfolding, these results show that the folding and unfolding domains include most of the same residues. However, three short segments change domains depending on whether the process is folding or unfolding. These changes are attributed to the very stable quaternary structure of rabbit muscle aldolase.     

Extended amino acid sequences around the active-site lysine residue of class-I fructose 1,6-bisphosphate aldolases from rabbit muscle, sturgeon muscle, trout muscle and ox liver.

1. Amino acid sequences covering the region between residues 173 and 248 [adopting the numbering system proposed by Lai, Nakai & Chang (1974) Science 183, 1204-1206] were derived for trout (Salmo trutta) muscle aldolase and for ox liver aldolase. A comparable sequence was derived for residues 180-248 of sturgeon (Acipenser transmontanus) muscle aldolase. The close homology with the rabbit muscle enzyme was used to align the peptides of the other aldolases from which the sequences were derived. The results also allowed a partial sequence for the N-terminal 39 residues for the ox liver enzyme to be deduced. 2. In the light of the strong homology evinced for these enzymes, a re-investigation of the amino acid sequence of rabbit muscle aldolase between residues 181 and 185 was undertaken. This indicated the presence of a hitherto unsuspected -Ile-Val-sequence between residues 181 and 182 and the need to invert the sequence -Glu-Val- to -Val-Glx- at positions 184 and 185. 3. Comparison of the available amino acid sequences of these enzymes suggested an early evolutionary divergence of the genes for muscle and liver aldolases. It was also consistent with other evidence that the central region of the primary structure of these enzymes (which includes the active-site lysine-227) forms part of a conserved folding domain in the protein subunit. 4. Detailed evidence for the amino acid sequences proposed has been deposited as Suy Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation

Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.     

Hybridization between fructose diphosphate aldolase subunits derived from diverse biological systems: anomolous hybridization behavior of some aldolase subunit types

In the present studies we investigated the abilities of fructose diphosphate aldolase subunits derived from diverse biological sources to form stable heterotetramers with each other in vitro. Aldolase C subunits isolated from chicken brain readily "hybridized" with aldolase subunits derived from lobster muscle and wheat germ following reversible acid dissociation of mixtures of these enzymes; however, appreciable amounts of stable heterotetramers containing chicken C subunits and aldolase subunits isolated from two other invertebrates (Ascaris and squid) were not produced under the same conditions. In contrast to the situation with chicken C subunits, aldolase B subunits isolated from rat liver did not "hybridize" appreciably with lobster muscle or wheat germ aldolase subunits. The present observations are not consistent with the hypothesis that the abilities of different aldolase subunit types to form heterotetramers in vitro is governed solely by the evolutionary relationships which exist between the organisms from which the enzymes are derived.     

Comparative evolutionary analysis of rDNA ITS regions in Drosophila

The internal transcribed spacer (ITS) of the ribosomal DNA is generally considered to be under low functional constraint, and it is therefore often treated as a typical nonfunctional spacer sequence. We have analyzed the ITS regions of five species from the Drosophila melanogaster subgroup, two Drosophila species from outside this group (D. pseudoobscura and D. virilis), as well as from the more distantly related dipteran fly Musca domestica. The sequence comparisons show a distinctive conservation/divergence pattern, indicating that some regions are more conserved than others. Moreover, secondary-structure calculations indicate several conserved structural elements within the ITS regions. On the other hand, a statistical test that allows us to estimate the fraction of sites that are not under selective constraint suggests that more than half of the spacer is apparently free to diverge and evolves with a rate that is close to the neutral rate of sequence evolution in Drosophila. The ITS sequences can be used to derive a molecular phylogeny for the species under study. We find that the ITS tree is largely in line with the so-far-known phylogeny of this group of species, with one difference. The species most distant within the D. melanogaster subgroup is D. yakuba, rather than D. orena, as is normally assumed.      

Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulation of enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase. Each G protein contains an alpha subunit that binds and hydrolyzes guanine nucleotides and interacts with beta gamma subunits and specific receptor and effector proteins. Amphipathic and secondary structure analysis of the primary sequences of five different alpha chains (bovine alpha s, alpha t1 and alpha t2, mouse alpha i, and rat alpha o) predicted the secondary structure of a composite alpha chain (alpha avg). The alpha chains contain four short regions of sequence homologous to regions in the GDP binding domain of bacterial elongation factor Tu (EF-Tu). Similarities between the predicted secondary structures of these regions in alpha avg and the known secondary structure of EF-Tu allowed us to construct a three-dimensional model of the GDP binding domain of alpha avg. Identification of the GDP binding domain of alpha avg defined three additional domains in the composite polypeptide. The first includes the amino terminal 41 residues of alpha avg, with a predicted amphipathic alpha helical structure; this domain may control binding of the alpha chains to the beta gamma complex. The second domain, containing predicted beta strands and alpha helices, several of which are strongly amphipathic, probably contains sequences responsible for interaction of alpha chains with effector enzymes. The predicted structure of the third domain, containing the carboxy terminal 100 amino acids, is predominantly beta sheet with an amphipathic alpha helix at the carboxy terminus. We propose that this domain is responsible for receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations.

The aldolase genes represent an ancient gene family with tissue-specific isozymic forms expressed only in vertebrates. The chromosomal locations of the aldolase genes provide insight into their tissue-specific and developmentally regulated expression and evolution. DNA probes for the human aldolase-A and -C genes and for an aldolase pseudogene were used to quantify and map the aldolase loci in the haploid human genome. Genomic hybridization of restriction fragments determined that all the aldolase genes exist in single copy in the haploid human genome. Spot-blot analysis of sorted chromosomes mapped human aldolase A to chromosome 16, aldolase C to chromosome 17, the pseudogene to chromosome 10; it previously had mapped the aldolase-B gene to chromosome 9. All loci are unlinked and located on to two pairs of morphologically similar chromosomes, a situation consistent with tetraploidization during isozymic and vertebrate evolution. Sequence comparisons of expressed and flanking regions support this conclusion. These locations on similar chromosome pairs correctly predicted that the aldolase pseudogene arose when sequences from the aldolase-A gene were inserted into the homologous aldolase location on chromosome 10.     

The primary structure of rat liver glycogen synthase deduced by cDNA cloning. Absence of phosphorylation sites 1a and 1b

The cDNA for rat liver glycogen synthase was isolated by screening a rat liver cDNA library constructed in lambda gt11. The cDNA was 2.4 kilobases in length and encoded a protein of 703 amino acid residues with a molecular mass of 80.5 kDa. Comparison of the rat liver and the human muscle sequences show that the amino- and carboxyl-terminal regions are quite divergent as compared to the internal sequences which show an 80% identity. The rat liver carboxyl-terminal region is truncated by 33 residues and has only 46% identity with the muscle sequence but retains the common feature of a low content of hydrophobic amino acids (13%). Phosphorylation sites 1a and 1b, which are the primary targets for phosphorylation by cAMP-dependent protein kinase, are absent in the liver sequence. The presence of these divergent, structurally anomalous carboxyl-terminal regions in liver and muscle glycogen synthase suggests the absence of the requirement that they possess a tertiary structure that is integral to that of the protein core. A model is proposed in which this region interacts with a catalytic core to maintain the I state, and in which phosphorylation serves to uncouple this interaction.     

Rat aldolase A messenger RNA: the nucleotide sequence and multiple mRNA species with different 5'-terminal regions

The nucleotide sequence of aldolase A mRNA in rat skeletal muscle was determined using recombinant cDNA clones and a cDNA synthesized by primer extension. The sequence is composed of 1343 nucleotides (nt) except for the poly(A) tail. Based on the sequence analysis we have deduced an open reading frame with 363 amino acids (aa) (Mr 39134). The sequence suggests several nt polymorphisms in the mRNA population, one of which causes an aa change. The determined sequence of rat aldolase A mRNA was compared with the published ones of rabbit aldolase A or rat aldolase B mRNAs. The homology between rat and rabbit aldolase A mRNA sequences is greater than that between rat aldolase A and B mRNA sequences. Multiple aldolase A mRNAs having different Mrs were detected in the various tissues, and appeared to be expressed in a tissue-specific manner. Further analysis suggests that differences in mRNA length are due to differences in the 5'-noncoding terminal region.     

Changes of aldolase A and B messenger RNA levels in rat liver during azo-dye-induced hepatocarcinogenesis

The expression of aldolase A and B mRNAs during azo-dye-induced carcinogenesis in rat liver was examined. After feeding the dye for 18 weeks, the level of aldolase A mRNA increased to about 11 times that in a normal liver, with the concomitant decrease of aldolase B mRNA level to about 25% of that in a normal liver. These changes did not occur progressively during the carcinogenesis, but occurred as an additional phase after 4 week-feeding of the azo-dye. At this stage, the levels of aldolase A and B mRNAs were about 7 times and 45% of that in a normal liver, respectively. This biphasic pattern in the aldolase isozyme expression in the azo-dye-fed rat liver is discussed together with the kinetic data of the enzyme activity.     

DNA methylation and the regulation of aldolase B gene expression

DNA methylation was studied as a potential factor for the regulation of tissue-specific and developmentally specific expression of the rat aldolase B gene. We examined cytosine methylation in the HpaII and HhaI recognition sequences in the aldolase B gene in aldolase expressing and nonexpressing tissues and cells. Out of the 15 methyl-sensitive restriction sites examined, the sites in the 3'-half and 3'-flanking regions were found to be heavily methylated in all the tissues or cells, regardless of the level of aldolase B gene expression. However, the methylation pattern in the region immediately upstream and in the 5'-half of the gene exhibited tissue-specificity: the site located about 0.13 kb upstream of the cap site (just next to the CCAAT box), and the sites in the first intron (intron 1) were heavily methylated in nonexpressing cells and tissues (ascites hepatoma AH130 and brain), whereas those in an expressing tissue (liver) were considerably less methylated. These results suggest that cytosine methylation at the specific sites in the 5'-flanking and 5'-half regions of the gene is associated with repression of the gene activity. However, the gene is still substantially methylated in the fetal liver on day 16 of gestation, when it is in a committed state for rapid activation in the period immediately afterwards (Numazaki et al. (1984) Eur. J. Biochem. 152, 165-170). This suggests that demethylation of the methylated cytosine residues in the specific gene region is not necessarily required before activation of the gene during development, but it may occur along with or after the activation.