The photosynthetic protist Euglena gracilis is one of few organisms known to possess both class-I and class-II fructose-1,6-bisphosphate aldolases (FBA). We have isolated cDNA clones encoding the precursor of chloroplast class-I FBA and cytosolic class-II FBA from Euglena. Chloroplast class-I FBA is encoded as a single subunit rather than as a polyprotein, its deduced transit peptide of 139 amino acids possesses structural motifs neccessary for precursor import across Euglena's three outer chloroplast membranes. Evolutionary analyses reveal that the class-I FBA of Euglena was recruited to the chloroplast independently from the chloroplast class-I FBA of chlorophytes and may derive from the cytosolic homologue of the secondary chlorophytic endosymbiont. Two distinct subfamilies of class-II FBA genes are shown to exist in eubacteria, which can be traced to an ancient gene duplication which occurred in the common ancestor of contemporary gram-positive and proteobacterial lineages. Subsequent duplications involving eubacterial class-II FBA genes resulted in functional specialization of the encoded products for substrates other than fructose-1,6-bisphosphate. Class-II FBA genes of Euglena and ascomycetes are shown to be of eubacterial origin, having been acquired via endosymbiotic gene transfer, probably from the antecedants of mitochondria. The data provide evidence for the chimaeric nature of eukaryotic genomes.

We report the sequences of full-length cDNAs for the nuclear genes encoding the chloroplastic and cytosolic fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) from spinach. A comparison of the deduced amino-acid sequences with one another and with published cytosolic aldolase sequences of other plants revealed that the two enzymes from spinach share only 54% homology on their amino acid level whereas the homology of the cytosolic enzyme of spinach with the known sequences of cytosolic aldolases of maize, rice and Arabidopsis range from 67 to 92%. The sequence of the chloroplastic enzyme includes a stroma-targeting N-terminal transit peptide of 46 amino acid residues for import into the chloroplast. The transit peptide exhibits essential features similar to other chloroplast transit peptides. Southern blot analysis implies that both spinach enzymes are encoded by single genes.

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l h e plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. l h e class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. l h e native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The FBP aldolase is one of the essential enzymes in glycolysis, gluconeogenesis, and the Calvin cycle. Two distinct classes of aldolases have been detected in biological systems (Richards and Rutter, 1961a;Rutter, 1964). Class I aldolases are tetrameric proteins with a molecular mass of about 160 kD. They form a Schiff's base with the substrate and are inactivated by borohydride (NaBH4) but not by metal-chelating agents (Rutter, 1964). Class I1 aldolases are dimers with a molecular mass of about 80 kD and form an endiole as reaction intermediate. The catalytic activity of the latter enzyme depends on divalent metal ions and is, therefore, inhibited by EDTA (Richards and Rutter, 1961b;Rutter, 1964). Class I aldolases are present in animals, higher plants, fems, mosses, and some eukaryotic algae. Class I1 aldolases are confined to more primitive organisms such as bacteria, cyanobacteria, and fungi. Only a few bacteria possess, in addition or exclusively, a class I aldolase. Algae and protists show a diverse distribution of class I and class I1 aldolases (Rutter, 1964;Antia, 1967;Lebherz and Rutter, 1969; Buko-* Correspondinz author; fax 49-30-838-4313. wiecki and Anderson, 1974; Altekar, 1986, 1989; for reviews see March and Lebherz, 1992;Schnarrenberger et al., 1992).Class I aldolases have been sequenced from several animals, protists, plants, and algae. The homology among the eukaryotic aldolases is at least 50% . Recently, the sequence of the class I aldolase of the prokaryote Staphylococcus carnosus was also determined. The latter enzyme differs from the eukaryotic aldolases because it is a monomer of somewhat smaller size (33 kD) than the eukaryotic class I aldolases. Its homology with the eukaryotic aldolases is only 30% (Witke and Gotz, 1993). Therefore, the S. carnosus aldolase has to be considered as ancestral in respect to both oligomeric state and sequence conservation.Class I1 aldolase sequences exist for the enzymes of yeast, Escherichia coli, and Co ynebacterium glutamicum (Alefounder et al., 1989;Schwellberger et al., 1989;von der Osten et al., 1989). They do not show any homology with class I aldolases. It was concluded that class I1 aldolases developed independently of class I aldolases in evolution (March and Lebherz, 1992). Further, the sequence homology among the class I1 aldolases is only on the order of 37 to 45%. This may indicate a greater variability among class I1 aldolases than among class I aldolases.Euglena gracilis is one of the few eukaryotic organisms that contains...

Genomic clones encoding the plastidic fructose-1,6-bisphosphate aldolase of Chlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of the C. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5' border sequences of introns in the aldolase gene of C. reinhardtii exhibit the conserved plant consensus sequence. The 3' acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in the C. reinhardtii genome.