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

摘要

Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA) is a member of the class II zinc-ndependent aldolase family that catalyzes the cleavage of D-fructose 1,6-bisphosphate (FBP) intondihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (G3P). In addition to the activensite zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme,nwhich when replaced with an alanine residue renders the enzyme inactive. A comparison of the crystalnstructures of D83A FBPA in complex with FBP and of wild-type FBPA in the unbound state revealed ansubstrate-induced conformational transition of loops in the vicinity of the active site and a shift in thenlocation of Zn2+. When FBP binds, the Zn2+ shifts up to 4.6 Å toward the catalytic Asp83, which bringsnthe metal within coordination distance of the Asp83 carboxylate group. In addition, the structure of wild-ntype FBPA was determined in complex with the competitive inhibitor D-tagatose 1,6-bisphosphate (TBP),na FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structurenof FBPA in complex with phosphoglycolohydroxamate, an analogue of the postulated DHAP ene-diolatenintermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orientnthe Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl groupnto the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote forncoordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novelnfeatures observed in the FBPA-FBP structure. The mechanism invokes coordination and coplanarity ofnthe Zn2+ with the FBP’s O-C(3)-C(4)-O group concomitant with coordination of the Asp83 carboxylicngroup. Catalysis is accompanied by movement of Zn2+ to a site coplanar with the O-C(2)-C(3)-O groupnof the DHAP. glFBPA exhibits strict substrate specificity toward FBP and does not cleave TBP. Thenactive sites of FBPAs contain an aspartate residue equivalent to Asp255 of glFBPA, whereas tagatose-n1,6-bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this asparticnacid is a likely determinant of FBP versus TBP specificity. Replacement of Asp255 with an alanine resultednin an enzyme that possesses double specificity, now cleaving TBP (albeit with low efficacy; kcat/Km ) 80nM-1ns-1n) while maintaining activity toward FBP at a 50-fold lower catalytic efficacy compared with thatnof wild-type FBPA. The collection of structures and sequence analyses highlighted additional residuesnthat may be involved in substrate discrimination.