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. 2000 Sep 15;350(Pt 3):823–828.

Functional and molecular modelling studies of two hereditary fructose intolerance-causing mutations at arginine 303 in human liver aldolase.

R Santamaria 1, G Esposito 1, L Vitagliano 1, V Race 1, I Paglionico 1, L Zancan 1, A Zagari 1, F Salvatore 1
PMCID: PMC1221316  PMID: 10970798

Abstract

We have identified a novel hereditary fructose intolerance mutation in the aldolase B gene (i.e. liver aldolase) that causes an arginine-to-glutamine substitution at residue 303 (Arg(303)-->Gln). We previously described another mutation (Arg(303)-->Trp) at the same residue. We have expressed the wild-type protein and the two mutated proteins and characterized their kinetic properties. The catalytic efficiency of protein Gln(303) is approx. 1/100 that of the wild-type for substrates fructose 1,6-bisphosphate and fructose 1-phosphate. The Trp(303) enzyme has a catalytic efficiency approx. 1/4800 that of the wild-type for fructose 1,6-bisphosphate; no activity was detected with fructose 1-phosphate. The mutation Arg(303)-->Trp thus substitution impairs enzyme activity more than Arg(303)-->Gln. Three-dimensional models of wild-type, Trp(303) and Gln(303) aldolase B generated by homology-modelling techniques suggest that, because of its larger size, tryptophan exerts a greater deranging effect than glutamine on the enzyme's three-dimensional structure. Our results show that the Arg(303)-->Gln substitution is a novel mutation causing hereditary fructose intolerance and provide a functional demonstration that Arg(303), a conserved residue in all vertebrate aldolases, has a dominant role in substrate binding during enzyme catalysis.

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Selected References

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  1. Ali M., Rellos P., Cox T. M. Hereditary fructose intolerance. J Med Genet. 1998 May;35(5):353–365. doi: 10.1136/jmg.35.5.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blom N., Sygusch J. Product binding and role of the C-terminal region in class I D-fructose 1,6-bisphosphate aldolase. Nat Struct Biol. 1997 Jan;4(1):36–39. doi: 10.1038/nsb0197-36. [DOI] [PubMed] [Google Scholar]
  3. Choi K. H., Mazurkie A. S., Morris A. J., Utheza D., Tolan D. R., Allen K. N. Structure of a fructose-1,6-bis(phosphate) aldolase liganded to its natural substrate in a cleavage-defective mutant at 2.3 A(,). Biochemistry. 1999 Sep 28;38(39):12655–12664. doi: 10.1021/bi9828371. [DOI] [PubMed] [Google Scholar]
  4. Cregut D., Serrano L. Molecular dynamics as a tool to detect protein foldability. A mutant of domain B1 of protein G with non-native secondary structure propensities. Protein Sci. 1999 Feb;8(2):271–282. doi: 10.1110/ps.8.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Doyle S. A., Tolan D. R. Characterization of recombinant human aldolase B and purification by metal chelate chromatography. Biochem Biophys Res Commun. 1995 Jan 26;206(3):902–908. doi: 10.1006/bbrc.1995.1128. [DOI] [PubMed] [Google Scholar]
  6. Gamblin S. J., Davies G. J., Grimes J. M., Jackson R. M., Littlechild J. A., Watson H. C. Activity and specificity of human aldolases. J Mol Biol. 1991 Jun 20;219(4):573–576. doi: 10.1016/0022-2836(91)90650-u. [DOI] [PubMed] [Google Scholar]
  7. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  8. Miller S. A., Dykes D. D., Polesky H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb 11;16(3):1215–1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Morris A. J., Tolan D. R. Site-directed mutagenesis identifies aspartate 33 as a previously unidentified critical residue in the catalytic mechanism of rabbit aldolase A. J Biol Chem. 1993 Jan 15;268(2):1095–1100. [PubMed] [Google Scholar]
  10. Paolella G., Santamaria R., Izzo P., Costanzo P., Salvatore F. Isolation and nucleotide sequence of a full-length cDNA coding for aldolase B from human liver. Nucleic Acids Res. 1984 Oct 11;12(19):7401–7410. doi: 10.1093/nar/12.19.7401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Salvatore F., Izzo P., Paolella G. Aldolase gene and protein families: structure, expression and pathophysiology. Horiz Biochem Biophys. 1986;8:611–665. [PubMed] [Google Scholar]
  12. Santamaria R., Scarano M. I., Esposito G., Chiandetti L., Izzo P., Salvatore F. The molecular basis of hereditary fructose intolerance in Italian children. Eur J Clin Chem Clin Biochem. 1993 Oct;31(10):675–678. doi: 10.1515/cclm.1993.31.10.675. [DOI] [PubMed] [Google Scholar]
  13. Santamaria R., Tamasi S., Del Piano G., Sebastio G., Andria G., Borrone C., Faldella G., Izzo P., Salvatore F. Molecular basis of hereditary fructose intolerance in Italy: identification of two novel mutations in the aldolase B gene. J Med Genet. 1996 Sep;33(9):786–788. doi: 10.1136/jmg.33.9.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Santamaria R., Vitagliano L., Tamasi S., Izzo P., Zancan L., Zagari A., Salvatore F. Novel six-nucleotide deletion in the hepatic fructose-1,6-bisphosphate aldolase gene in a patient with hereditary fructose intolerance and enzyme structure-function implications. Eur J Hum Genet. 1999 May-Jun;7(4):409–414. doi: 10.1038/sj.ejhg.5200299. [DOI] [PubMed] [Google Scholar]
  15. Wang J., Morris A. J., Tolan D. R., Pagliaro L. The molecular nature of the F-actin binding activity of aldolase revealed with site-directed mutants. J Biol Chem. 1996 Mar 22;271(12):6861–6865. [PubMed] [Google Scholar]

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