Long-term ingestion has effects on diabetes and obesity. The most drastic and common genetic disorder of fructose metabolism is hereditary fructose intolerance (HFI). Lack of knowledge about sites of fructose metabolism and about genotype-phenotype relationships still exists for this disease and reflects the incomplete understanding of normal fructose metabolism. Answers to two major questions will provide new information. First, other than liver and kidney, what other tissues play a role in fructose metabolism? Second, can small molecules be found that stabilize the major defective enzyme in HFI, that harboring an A149P substitution (AP-aldolase)? The proposed investigations will, 1) define sites for fructose assimilation and utilization using a combination of bioinformatics and molecular approaches, 2) determine a high-resolution structure of AP-aldolase, and use it to find stabilizing small-molecule ligands by both structure-based ligand design (SBLD) and high-throughput screening of chemical libraries, 3) create animal models for HFI using gene-targeting techniques, and 4) identify HFI mutations in the diverse US population, in particular Hispanic, African-American, and other ethnic groups that have not been well characterized, and correlate these findings to any specific phenotypes in these ethnic groups. The large database of expressed sequence tags (dbEST) will be analyzed for overlapping expression profiles of the GLUT5, GLUT2, ketohexokinase, aldolase, hexokinase, and triose kinase in both mouse and humans to predict alternative sites of fructose metabolism. Verification and characterization of these global predictions will be done by quantitative reverse-transcriptase polymerase chain reaction (Q-PCR), RNA in situ hybridization (RISH), and identification of metabolic intermediates during the oxidation of radioactive fructose. For the second hypothesis, a high-resolution structure of AP-aldolase will be determined by macromolecular X-ray crystallography. Screening large libraries of small molecules (produced by combinatorial chemistry) using a thermal-stability assay of AP-aldolase in conjunction with SBLD will identify small molecules that restore enzyme function. Should the gene-targeted animal model mimic the human HFI pathology, the metabolic profiles and sites of fructose metabolism will be determined. Should the animal model be asymptomatic, differences in the ability to metabolize fructose will be compared to previously determined sites and pathways for fructose metabolism in normal mice and humans. Lastly, blood samples from African-American and Hispanic-American HFI subjects will be used for identification of gene defects by direct DNA sequencing, thus offering a reliable non-invasive diagnostic method to these Americans. ? ?

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-GTIE (90))
Program Officer
Mckeon, Catherine T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Boston University
Schools of Arts and Sciences
United States
Zip Code
Oppelt, Sarah A; Zhang, Wanming; Tolan, Dean R (2017) Specific regions of the brain are capable of fructose metabolism. Brain Res 1657:312-322
Stopa, Jack D; Chandani, Sushil; Tolan, Dean R (2011) Stabilization of the predominant disease-causing aldolase variant (A149P) with zwitterionic osmolytes. Biochemistry 50:663-71
Funari, Vincent A; Voevodski, Konstantin; Leyfer, Dimitry et al. (2010) Quantitative gene expression profiles in real time from expressed sequence tag databases. Gene Expr 14:321-36
Coffee, Erin M; Yerkes, Laura; Ewen, Elizabeth P et al. (2010) Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population. J Inherit Metab Dis 33:33-42
Coffee, Erin M; Tolan, Dean R (2010) Mutations in the promoter region of the aldolase B gene that cause hereditary fructose intolerance. J Inherit Metab Dis 33:715-25
Pezza, John A; Stopa, Jack D; Brunyak, Elizabeth M et al. (2007) Thermodynamic analysis shows conformational coupling and dynamics confer substrate specificity in fructose-1,6-bisphosphate aldolase. Biochemistry 46:13010-8
Funari, Vincent A; Crandall, James E; Tolan, Dean R (2007) Fructose metabolism in the cerebellum. Cerebellum 6:130-40
Choi, Kyung H; Lai, Vicky; Foster, Christine E et al. (2006) New superfamily members identified for Schiff-base enzymes based on verification of catalytically essential residues. Biochemistry 45:8546-55
Hopkins, Christopher E; Hernandez, Gonzalo; Lee, Jonathan P et al. (2005) Aminoethylation in model peptides reveals conditions for maximizing thiol specificity. Arch Biochem Biophys 443:1-10
Malay, Ali D; Allen, Karen N; Tolan, Dean R (2005) Structure of the thermolabile mutant aldolase B, A149P: molecular basis of hereditary fructose intolerance. J Mol Biol 347:135-44

Showing the most recent 10 out of 11 publications