tolan@bu.edu

Cell Signaling and Gene Regulation Biochemistry Genetics Molecular Ecology and Evolution

Professor of Biology
Ph.D., University of California, 1981

Biochemistry, enzymology, molecular & human genetics, evolution, and developmental biology of aldolases

Virtually all organisms contain the glycolytic enzyme fructose bisphosphate aldolase. Vertebrates have three distinguishable isoforms of the enzyme; aldolase A, B, and C. Each of these has different tissue distribution and catalytic properties that appropriately reflect the role of each isozyme in sugar metabolism in the tissue(s) where they are expressed. During vertebrate fetal development, the tissue-specific expression of each isozyme is controlled by distinct changes in gene expression. The aldolases have also been implicated in a number of moonlighting functions involved in cell motility, infection, insulin signaling, and endocytosis. It is the goal of the laboratory to understand the biochemical, evolutionary, and developmental mechanisms responsible for the distinguishing features of the various aldolases. Techniques in enzymology, spectroscopy, x-ray crystallography, genetics, gene-targeting, cell culture, RNA interference, and molecular evolution are being utilized to address these questions.

Structure and function of aldolases - The structural and functional details of this ancient and essential glycolytic enzyme are being investigated using site-directed mutagenesis of catalytically important residues coupled with steady-state and single-turnover kinetic analysis of the partial reactions in the multi-step catalytic mechanism. These are complimented with structural analysis via x-ray crystallography and cryo-electron microscopy of the structures of the different isozymes complexed with substrate or inhibitor. Stopped-flow spectroscopy and rapid quench techniques are used to assess the role of catalytically important residues in the enzyme mechanism. The vertebrate aldolases, are also being used as a model for studies on the nature and role of quaternary structure in proteins.

Characterization of the genetic defects in hereditary fructose intolerance (HFI) - Insight into the structure and function of one isozyme found in the liver, aldolase B, can be gained by the study of inborn errors of metabolism. Mutations in the aldolase B gene manifest themselves in HFI, a genetic disorder that can cause liver disease, coma, and death. It is especially problematic for newborn infants at the time they are weaned to fructose-containing foods. We are investigating the mutations in the human aldolase B gene from families with HFI using a combination of techniques including the polymerase chain reaction (PCR), allele specific oligonucleotide (ASO) hybridization, and automated DNA cycle-sequencing. Current projects include; construction of an HFI animal model via knock-out mouse technology, determination of the number and types of different mutations in known HFI patients from different ethnic groups to aid in HFI diagnosis, and characterization of the mutant enzymes. This knowledge will lead to a better understanding of enzyme function, provide a correlation of the particular enzyme defect to the clinical findings, and be useful for newborn screening and genetic counseling.

Evolution of the aldolases - The evolutionary history of the aldolase isozymes and their adaptation to their present physiological roles offer powerful insights into the structure and function of the enzyme as well as cellular functions. Current projects include the characterization of moonlighting functions that have evolved by oblating these functions with RNAi and mutant forms of the enzyme in tissue culture cells. In addition, the isolation and characterization of aldolases from urochordates, fish, and other non-mammalian vertebrates, and the phylogenetic analysis of vertebrate aldolase sequences are being used to determine of the number of aldolase genes and their relationship to each other in non-mammalian species.

Funari, V.A., Voevodski, K., Leyfer, D., Yerkes, L.L., Cramer, D., and Tolan, D.R. (2008) Virtual Northern Blot (VNB): an algorithm for quantitative expression profiles from expressed sequence tag database. BMC-Bioinformatics, submitted

Sherawat, M. Tolan, D.R., and Allen, K.N. (2008) Crystal structure of a rabbit muscle fructose-1, 6-bisphosphate aldolase A dimer variant with dihydroxyacetone phosphate. Acta Crystallographica C, in press

Pezza, J.A., Stopa J.D., Brunyak E.M., Allen, K.N. and Tolan, D.R. (2007) Thermodynamic analysis shows conformational coupling and dynamics confer substrate specificity in fructose-1,6-bisphosphate aldolase. Biochemistry. Nov 13;46(45):13010-8. Epub 2007 Oct 13.
[PMID: 17935305]

Pezza, J.A., Allen, K.N. and Tolan, D.R. (2006) Coupling interactions reveal that isozyme-specific residues confer substrate specificity in fructose-1,6-bisphophate aldolase. Proc. Natl. Acad. Sci USA, submitted

Funari, V.A., Crandall, J.E. and Tolan, D.R. (2007) Fructose Metabolism in the Cerebellum. 6(2):130-40. [PMID: 17510913]

Choi, K.-H., Lai, V., Foster, C.E., Morris, A.J., Tolan, D.R. and Allen, K.N. 2006. New Superfamily Members Identified for Schiff-base Enzymes Based on Verification of Catalytically Essential Residues. Biochemistry. Jul 18;45(28):8546-55.

Malay, A.D., Allen, K.N. and Tolan, D.R. 2005.   Structure of the thermolabile mutant aldolase B, A149P:   molecular basis of hereditary fructose intolerance. J Mol Biol.   Mar 18;347(1):135-44.

Canete-Soler, R., Reddy, K.S., Tolan, D.R. and Zhai, J. 2005. Aldolases A and C are ribonucleolytic components of a neuronal complex that regulates the stability of the light-neurofilament (NF-L) mRNA. J Neurosci. Apr 27;25(17):4353-64.  

Funari, V.A., Herrera, V.L.M., Freeman, D. and Tolan, D.R. 2005. Genes required for fructose metabolism are expressed in Purkinje cells in the cerebellum.   Brain Res Mol Brain Res. Dec 14;142(2):115-22.

Stormon, M.O., Cutz, E., Furuya, K., Bedford, M., Yerkes, L., Tolan, D.R. and Feigenbaum, A. 2004. A six-month-old infant with liver steatosis. J Pediatr. Feb;144(2):258-63.

Choi, K.-H. and Tolan, D.R. 2004. Presteady-state kinetic evidence for a ring-opening activity in fructose-1,6-(bis)phosphate aldolase. J Am Chem Soc. Mar 24;126(11):3402-3.

Arakaki, T.L., Pezza, J.A., Cronin, M.A., Hopkins, C.E., Zimmer, D.B., Tolan D.R. and Allen, K.N.   2004. Structure of human brain fructose 1,6-bisphosphate aldolase:   Linking isozyme structure with function. Protein Sci. Dec;13(12):3077-84.  

Pezza JA, Choi KH, Berardini TZ, Beernink PT, Allen KN, Tolan DR. 2003. Spatial clustering of isozyme-specific residues reveals unlikely determinants of isozyme specificity in fructose-1,6-bisphosphate aldolase. J Biol Chem. May 9;278(19):17307-13.

Tolan, D.R., Schuler, B., Beernink, P.T. and Jaenicke, R.   2003. Thermodynamic analysis of the dissociation of the aldolase tetramer substituted at one or both of the subunit interfaces. Biol Chem. Oct-Nov;384(10-11):1463-71.

Tolan, D.R. 1995. Molecular basis of hereditary fructose intolerance: Mutations and polymorphisms in the human aldolase B gene. Human Mutation 6:210-218.

Questions and comments are always welcome.
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This document was last modified on February 12, 2008.