Crystal Structure of an Archaeal Class I Aldolase and the Evolution of (βα)8 Barrel Proteins

Abstract

Fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in the glycolytic pathway. FBPAs from archaeal organisms have recently been identified and characterized as a divergent family of proteins. Here, we report the first crystal structure of an archaeal FBPA at 1.9-Å resolution. The structure of this 280-kDa protein complex was determined using single wavelength anomalous dispersion followed by 10-fold non-crystallographic symmetry averaging and refined to an R-factor of 14.9% (R free 17.9%). The protein forms a dimer of pentamers, consisting of subunits adopting the ubiquitous (␤␣) 8 barrel fold. Additionally, a crystal structure of the archaeal FBPA covalently bound to dihydroxyacetone phosphate was solved at 2.1-Å resolution. Comparison of the active site residues with those of classical FBPAs, which share no significant sequence identity but display the same overall fold, reveals a common ancestry between these two families of FBPAs. Structural comparisons, furthermore, establish an evolutionary link to the triosephosphate isomerases, a superfamily hitherto considered independent from the superfamily of aldolases.The fructose-1,6-bisphosphate aldolase (FBPA, 1 EC 4.1.2.13) fulfills an important function in the reversible Embden-Meyerhof-Parnas pathway and the Calvin cycle. The enzyme catalyzes the reversible aldol condensation of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Two different types of FBPA, termed class I and II (FBPA I and FBPA II), with different enzymatic mechanisms have so far been discovered. Whereas class I enzymes utilize the ⑀-amino group of the active site lysine to form a Schiff base with the substrate, the FBPAs II use divalent metal ions to stabilize the intermediate (1). The classical FBPA I is mainly found in eukaryotes (e.g. animals, higher plants, protozoa, algae, and yeast) and is only identified in a few bacteria. FBPA II is in contrast mainly used by bacteria (2). Although both classes of enzymes adopt the (␤␣) 8 -(TIM)-barrel fold, their insignificant overall sequence similarity casts doubts on whether they share a common ancestor. However, more recent analyses based on sequence, structure, and functional information suggest that both FBPA classes share a common evolutionary origin (3, 4).The recently identified archaeal FBPA (FBPA IA) represents a third type of aldolase. Archaeal organisms appear to rely solely on the archaeal class I enzyme for FBPA activity (5). Although these enzymes use the Schiff base mechanism, they do not show any significant overall sequence similarity to classical FBPA I. Some bacterial genomes contain FBPA IA homologs (6) in addition to FBPA II, the typical bacterial FBPA (e.g. Escherichia coli) (2). So far no eukaryotic homologs of the archaeal class I enzymes are known.Biochemical analyses of archaeal FBPA from Thermoproteus tenax and Pyrococcus furiosus reveal that this enzyme, like the classical FBPA I, catalyzes th...

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

“…The C-terminal region (residues 343-363) is conformationally mobile (12,13), has an extended secondary structure (14,15), and distinguishes mammalian aldo-lases and their orthologues. Although a number of structural studies have been performed that have investigated intermediates in class I aldolases (15)(16)(17)(18), characterization of the interconversion between the iminium and the enamine has so far proven elusive at the structural level.…”

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of fructose-1,6-bisphosphate aldolase fromEscherichia coli

“…over, the asymmetric unit of FBPA I from known archaeal enzyme structures contains ten molecules (Lorentzen et al, 2003(Lorentzen et al, , 2005Siebers et al, 2001). Preliminary crystallographic research on Ec-FBPA I will facilitate future structural and functional studies.…”

“…Escherichia coli is one of the few organisms which contain both types of FBPA (Thomson et al, 1998). The FBPA I from E. coli (Ec-FBPA I) has a maximum of only 27% amino-acid sequence identity to FBPA I enzymes of known structure (Lorentzen et al, 2003). In this communication, the 2.0 Å resolution crystal structure of Ec-FBPA I is reported, which will enable detailed structural analysis of its key components.…”

“…The crystallographic study of FBPA I can be traced back to 1987 (Sygusch et al, 1987), since when more than 30 FBPA I structures have been deposited in the PDB. Several structures of FBPA I from eukaryotes and archaea (Hester et al, 1991;Lorentzen et al, 2003Lorentzen et al, , 2005Galkin et al, 2009;Blom & Sygusch, 1997) have been solved, but there is no information about the three-dimensional structures of bacterial FBPA I enzymes. Escherichia coli is one of the few organisms which contain both types of FBPA (Thomson et al, 1998).…”

Modelling bound ligands in protein crystal structures

“…The location of the DHAP molecule in the difference map of an aldolase structure (PDB code 1ok4; Lorentzen et al, 2003) was determined using the clustering algorithm around the residues where the ligand was known to bind a priori. The re®ned interpretation is shown in Fig.…”