Structural Insights into the Substrate Binding and Stereoselectivity of Giardia Fructose-1,6-bisphosphate Aldolase (original) (raw)

Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA)1 is a member of the Class II zincdependent aldolase family that catalyzes the cleavage of D-fructose-1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced by an alanine residue renders the enzyme inactive. A comparison of the crystal structures of the D83A FBPA in complex with FBP and of the wild-type FBPA in the unbound state revealed a substrate induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn 2+. Upon FBP binding, the Zn 2+ shifts up to 4.6 Å towards the catalytic Asp83, which brings the metal within coordination distance to the Asp83 carboxylate group. In addition, the structure of wild-type FBPA was determined in complex with the competitive inhibitor D-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analog of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA/FBP structure. The mechanism invokes coordination and co-planarity of the Zn 2+ with the FBP's O-C(3)-C(4)-O concomitant with coordination of Asp83 carboxylic group. Catalysis is accompanied by movement of Zn 2+ to a site co-planar with the O-C(2)-C(3)-O of the DHAP. glFBPA exhibit strict substrate specificity towards FBP and does not cleave TBP. The active sites of FBPAs contain an aspartate residue equivalent to Asp255 of glFBPA, whereas tagatose-1,6bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this aspartic acid is a likely determinant of FBP vs. TBP specificity. Replacement of Asp255 by an alanine resulted in an enzyme that possesses double specificity, now cleaving TBP (albeit with low efficacy; k cat /K m = 80 M −1 s −1) while maintaining activity towards FBP at 50-fold lower catalytic efficacy