Structural insights into the substrate binding and stereoselectivity of giardia fructose-1,6-bisphosphate aldolase

Abstract

Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA) is a member of the class II zinc-dependent 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 with an alanine residue renders the enzyme inactive. A comparison of the crystal structures of D83A FBPA in complex with FBP and of 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+). When FBP binds, the Zn(2+) shifts up to 4.6 A toward the catalytic Asp83, which brings the metal within coordination distance of 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 analogue 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 coplanarity of the Zn(2+) with the FBP's O-C(3)-C(4)-O group concomitant with coordination of the Asp83 carboxylic group. Catalysis is accompanied by movement of Zn(2+) to a site coplanar with the O-C(2)-C(3)-O group of the DHAP. glFBPA exhibits strict substrate specificity toward FBP and does not cleave TBP. The active sites of FBPAs contain an aspartate residue equivalent to Asp255 of glFBPA, whereas tagatose-1,6-bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this aspartic acid is a likely determinant of FBP versus TBP specificity. Replacement of Asp255 with 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 toward FBP at a 50-fold lower catalytic efficacy compared with that of wild-type FBPA. The collection of structures and sequence analyses highlighted additional residues that may be involved in substrate discrimination.

Figure 1

(A) The reaction catalyzed by fructose-1,6-bisphosphate aldolase (FBPA). (B) D-tagatose-1,6-bisphosphate (TBP), a C(4) hydroxyl epimer of D-fructose-1,6-bisphosphate (FBP). (C) phosphoglycolohydroxamate (PGH), a DHAP ene-diolate transition-state analog.

Figure 2

Stereoscopic view of the electron density map in the vicinity of the active site. Difference Fourier electron density maps with the coefficients F_o−F_c and calculated phases generated omitting the ligands and zinc co-factors from the models. The maps are countered at 3σ level. (A & B) The apo structure contains sulfate ions and each subunit of the dimer exhibits different Zn²⁺ environment. Data resolution: 2.9 Ǻ (C) Bound TBP. Data resolution: 1.8 Ǻ (D) Bound FBP. Data resolution: 2.0 Ǻ

Figure 3

Stereoscopic representation of glFBPA in the unbound and ligand-bound states. The superposed molecules are depicted in blue (unbound) and pink (bound state) colors where the trace of the polypeptide chain is similar in both structures. The major differences occur in two loop regions: The terminal fragments of the two disordered loops of the apo glFBPA structure are highlighted in blue, and the same loops of the glFBPA/FBP structure are highlighted in red. The FBP is depicted as green stick model.

Figure 4

Binding of FBP (A) and TBP (B) to glFBPA. Stereoscopic view of the environment around the active site. Atomic colors are as follows: oxygen, red; nitrogen, blue; carbon (protein), gray; carbon (ligand), green; phosphor, orange; and zinc, steel blue. Key electrostatic interactions of the ligands are shown in dashed lines.

Figure 5

Local active site conformational transition associated with FBP binding to glFBPA (with a model of Asp83 based on other wild-type glFBPA structures). The Zn²⁺ position shifts by 4.6 Å towards the catalytic Asp83, which changes its coordination so that in the substrate cleavage mode (Zn2B) the Asp83 is coordinated to Zn²⁺. The Zn²⁺ and carbon atoms of the apo enzyme are colored in cyan. Other atomic colors are the same as in Figure 4. Note that His178 is disordered in the apo structure

Figure 6

Proposed catalytic mechanism of class II FBPA. The FBP C(4) position, the chirality of which distinguishes FBP from TBP, is indicated by an asterisk. Carbon numbering from 1 to 6 is maintained also after bond cleavage for clarity.

Figure 7

Sequence logos (27) of multiple sequence alignment for three regions of the FBPA and TBPA subfamilies. The overall height of the stack indicates the level of sequence conservation at each position and the height of symbols within the stack indicates the relative frequency of the particular amino acid at the position. The glFBPA numbering was used and every other amino acid residue is numbered.