Fructose-1,6-bisphosphate aldolase de classe II : aspects structural et dynamique dans le mécanisme réactionnel

Fructose-1,6-bisphosphate aldolase catalyzes the reversible aldol reaction in glucose metabolism interconverting dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (G3P) into D-fructose 1,6-bisphosphate (FBP). Aldolases are furthermore classified based on their reaction mechanism: class I aldolase (e. g. human aldolase) forms a covalent Schiff base intermediate with substrate, whereas class II aldolase utilizes a divalent metal cation in catalysis.  Class II aldolase is commonly found in pathogenic organisms such as Mycobacterium tuberculosis (tuberculosis), Giardia lamblia (giardiasis), Escherichia coli (diverse infections) and  Helicobacter pylori (ulcer and gastric cancer) but not in mammals. This distribution makes class II aldolase a potential target for drug discovery. Structure driven drug design depends on an explicit knowledge of the reaction mechanism of class II aldolase and its three-dimensional structure. Our current knowledge is lacking; existing aldolase crystal structures with reaction intermediates and with competitive inhibitors are not coherent with proposed mechanisms in literature.   The present study focuses on the catalytic role of two residues, each located on a mobile loop of  H. pylori class II aldolase and each implicated in a critical proton transfer step. Single mutants H180Q and E142A were characterized enzymatically and crystallized for X-ray structure determination. Crystal structures of reaction intermediates formed with substrate were determined. The catalytic mechanism requires proton abstraction at the FBP C4 hydroxyl group to initiate C3-C4 bond cleavage, first step of the retroaldol reaction. Our data supports His180 situated on the mobile loop beta6-alpha8, as the residue responsible for this proton transfer. Notably, His180 chelates the zinc ion in the native structure. The structural change induced due to C1 phosphate binding of FBP releases His180 to promote cleavage. Displacement of the catalytic zinc ion ensues, facilitating substrate binding and subsequent stabilization of the enediolate intermediate. Our results do not support the previous hypothesis of a catalytic role for Asp82 in C4 hydroxyl group proton abstraction; it rather plays an important role in maintaining structural integrity for active site binding. Displacement of the nascent aldehyde G3P and concomitant stereospecific protonation of the enediolate species generates the obligate triose phosphate, DHAP. Dissociation of DHAP from the active site completes the catalytic cycle. The residue responsible for initiating enediolate protonation was identified as residue Glu142, situated on mobile loop beta5-alpha7, and this is in agreement with previous kinetic studies of enediolate protonation in other class II aldolases, attributing the same role to this conserved residue. We devised a molecular dynamic simulation method to follow the catalytic loop folding event, further investigating details of the role of Glu142 in catalysis. We gained further knowledge of the cleavage event, although work remains to elucidate missing details of the catalysis and integrate our findings. By attributing a role in catalysis to loop beta6-alpha8 not limited to substrate binding, this loop of class II aldolases becomes a potential target in drug design. In addition, ligand independent zinc ion migration suggest it is possible to chelate the metal and restrain it far from the active site.

Keyphrases: Aldol reaction, Retro-aldol reaction, Proton transfer, Metalloenzyme, Crystallography, X-ray diffraction, Molecular dynamics