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Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase.

ABSTRACT: Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn(2+) binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K(i)=14?M) that is comparable to that of FBP (K(m)=2?M) or its inert analog TBP (K(i)=1?M). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3Å) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn(2+). The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn(2+) are not consistent with a strong interaction. To determine if Zn(2+)coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn(2+) coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn(2+) coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn(2+) coordination to binding of 8 to GlFBPA.

SUBMITTER: Li Z

PROVIDER: S-EPMC3071891 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase.

Li Zhimin Z Liu Zhengang Z Cho Dae Won DW Zou Jiwen J Gong Maozhen M Breece Robert M RM Galkin Andrey A Li Ling L Zhao Hong H Maestas Gabriel D GD Tierney David L DL Herzberg Osnat O Dunaway-Mariano Debra D Mariano Patrick S PS

Journal of inorganic biochemistry 20101230 4

Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn(2+) binding group. The inhibition activities for the GlFBPA ...[more]

PMID: 21333622

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Project description: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.

Dataset Information

Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase.

Publications

Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase.

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