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...