STRUCTURAL BASIS FOR SUBSTRATE RECOGNITION IN CLASS I TAGATOSE-1,6-BISPHOSPHATE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Class I glycolytic aldolases are enzymes that reversibly catalyze the cleavage of a biphosphorylated hexose, which yields glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP), after forming a protonated imine with a lysine residue. While the Tagatose-1,6-Bisphosphate Aldolase (TBPA) cleaves the tagatose-1,6-bisphosphate (TBP) and the fructose-1,6-bisphosphate (FBP) with similar affinities, the fructose-1,6-Bisphophate Aldolase (FBPA) is exclusively specific toward FBP. The cleavage/condensation products, differing only with regards as to stereochemical configuration of the third and fourth carbon atoms, raises the question of how aldolases stereospecifically recognize their cognate substrates. The rabbit muscle FBPA is well characterized, both kinetically and structurally. Moreover, an imine intermediate, trapped into the active site of the crystalline FBPA recently allowed the unveiling of significant aspects of the stereospecificity of this enzyme. However, the structural features of the permissive specificity of TBPA remain unknown. We have thus undertaken an x-ray crystallographic study of catalytic intermediates of the TBPA to reveal the molecular architecture of its active site and the residues involved in ligand recognition and catalysis. To this end, we have purified and crystallized native TBPA from Streptococcus pyogenes and its selenomethionylated derivative. MAD phasing experiments have allowed the determination of the tertiary structure of the enzyme revealing an (¿}¿¿/¿}¿¿)8 barrel subunit fold. We propose to determine the structures of reaction complexes formed with TBPA to high resolution to gain structural insight into the degenerate recognition by TBPA. Preliminary data obtained from crystal soaking experiemnts conducted in presence of substrate and their analogues show the feasibility of cryotrapping reaction intermediates in the TBPA active site. In order to unambiguously distinguish the steric configurations of cryotrapped reaction intermediates requires that we plan to collect diffraction data to at least 2A resolution. Data collection at X25 using the ADSC 3x3 detector has allowed us to collect such data to maximum resolution on native crystals otherwise not possible because of a long unit cell dimension in our crystals.