Research
Publications

Our research interests center on the structure and function of enzymes and the design and synthesis of enzyme inhibitors or activators with application to problems of medical relevance. Our focus is on enzymes which utilize tetrahydrofolates or the chemically related tetrahydrobiopterin as cofactors. In contrast to tetrahydrofolates which are synthesized from the vitamin, folic acid, tetrahydrobiopterin is synthesized in the cells in which it is utilized by three specific enzymes starting from GTP.

Neurotransmitter Biosynthesis
Tetrahydrobiopterin is an essential cofactor for the three aromatic amino acid hydroxylases, phenylalanine hydroxylase which catalyzes the first step in the catabolism of excess dietary phenylalanine, and tryptophan and tyrosine hydroxylases which catalyze the first and rate limiting steps in the biosynthesis of serotonin and the catecholamine neurotransmitters and hormones, respectively. Several neurological disorders, including Parkinson's disease and dystonia, are associated with decreased levels of tetrahydrobiopterin. Studies now in progress will allow optimization of the design of drugs for cofactor replacement therapy.

Regulation of Blood Pressure
Tetrahydrobiopterin is also essential for the production of nitric oxide. Nitric oxide (NO) is involved in a wide range of biological systems including regulation of blood pressure, neurotransmission and the immune system. NO is produced by three different isoforms of NO synthase, two of which are constitutive and one which is inducible. Over production of NO by the inducible form of NO synthase (iNOS) is believed to play a role in a variety of disease states, including sepsis, diabetes, arthritis and glomerular nephritis. When iNOS is induced there is a necessary co-induction of GTP cyclohydrolase, the rate limiting enzyme in BH4 biosynthesis. One project in our laboratory is the design and synthesis of inhibitors of GTP cyclohydrolase. A highly specific inhibitor of GTP cyclohydrolase would prevent the over-production of NO by limiting the availability of BH4. An inhibitor selective for iNOS would be invaluable in delineating the role of NO in the physiology or pathophysiology of a biological system, as well as having therapeutic potential for the treatment of
diseases mediated by excessive short term production of NO, such as septic shock.

Dehydratase/DCoH and Diabetes
Dehydratase/DCoH is a bifunctional protein which performs both catalytic and regulatory functions. In the cytoplasm as dehydratase it catalyzes the dehydration of 4a-hydroxy-tetrahydrobiopterin in the regeneration of tetrahydrobiopterin, an essential step in the reactions of the three aromatic amino acid hydroxylases. In the nucleus as DCoH (dimerization cofactor of hepatic nuclear factor 1a) it regulates dimerization of Hepatic nuclear transcription factor 1a (HNF1a). HNF1a is a transcription regulator for many proteins not only in liver and kidney, but also in the pancreas. Recently, it has been discovered that the source of one of the most common forms of maturity-onset diabetes of the young (MODY3) is a defect in the HNF1a gene. The way in which the mutant HNF1a causes this non-insulin dependent diabetes is not known. By studying the structure and function of DCoH we hope to determine the mechanism by which it is regulated to enable interaction with HNF1a. In order to find a link between the catalytic and transcriptional activities of dehydratase/DCoH, amino acid residues involved in dehydratase and DCoH function are being identified by site-directed mutagenesis.

1. The Influence of Side-Chain Chirality on the Activity and Regulation of Tyrosine and Phenylalanine Hydroxylases. S.W. Bailey, R.Y Chandrasekaran, S.B. Dillard and J.E. Ayling, in "Chemistry and Biology of Pteridines." Eds. H-Ch. Curtius, S. Ghisla and N. Blau. de Gruyter, Berlin, 625-655 (1990).

2. Why is the Cofactor for the Tetrahydrobiopterin Dependent Monooxygenases Not a Dihydroflavin? J.E. Ayling and S.W. Bailey, in "Biological Oxidation Systems." Eds. C.C. Reddy, G.A. Hamilton and K.M. Madyastha. Academic Press, N.Y., Vol. 1, 221-236 (1990).

3. The Kinetics and Regulation of Aromatic Amino Acid Hydroxylases: The Effect of Cofactor Structure. S.W. Bailey, S.B. Dillard, R.Y. Chandrasekaran, and J.E. Ayling, in "Biological Oxidation Systems." Eds. C.C. Reddy, G.A. Hamilton and K.M. Madyastha. Academic Press, N.Y., Vol. 1, 257-274 (1990).

4. Reaction of (6S)-Tetrahydrobiopterin With Phosphorylated and Unphosphorylated Tyrosine Hydroxylase. J.E. Ayling, S.B. Dillard, and S.W. Bailey, in "Pterins and Biogenic Amines in Neurology, Pediatrics ad Immunology." Eds. N. Blau, H-Ch. Curtius, R.A. Levine and R.G.H. Cotton. Lakeshore Publ., MI, 269-282 (1991).

5. The Cofactor Dependent Interaction of Molecular Oxygen with Phenylalanine Hydroxylase. S.W. Bailey, J.P. Crow, and J.E. Ayling, in "Flavins and Flavoproteins." Eds. B. Curti, S. Ronchi and G. Zanetti. Walter de Gruyter, Berlin, 247-250 (1991).

6. The Role of C6-Chirality of Tetrahydropterin Cofactor in Catalysis and Regulation of Tyrosine and Phenylalanine Hydroxylases. S.W. Bailey, S.B. Dillard and J.E. Ayling. Biochemistry 30, 10226-10235 (1991).

7. Synthesis of Tetrahydropteridine C6-Stereoisomers Including N5-Formyl-Tetrahydrofolic Acid. S.W. Bailey, R.Y. Chandrasekaran and J.E. Ayling. J. Org. Chem. 57, 4470-4477 (1992).

8. "Chemistry and Biology of Pteridines and Folates", Eds. J.E. Ayling, M.G. Nair and C.M. Baugh, Advances in Experimental Medicine and Biology, Volume 338 (1993).

9. The Mechanism of Cofactor Regeneration During Phenylalanine Hydroxylation. S.W. Bailey, S.R. Boerth, S.B. Dillard and J.E. Ayling. Adv. Exp. Med. Biol. 338, 47-54 (1993).

10. Catalytic Characterization of 4a-Hydroxy-tetrahydropterin Dehydratase. I. Rebrin, S.W. Bailey, S.R. Boerth, M.D. Ardell and J.E. Ayling. Biochemistry 34, 5801-5810 (1995).

11. Synthesis of 4a-Hydroxy-tetrahydropterins and the Mechanism of their Non-enzymatic Dehydration to Quinoid Dihydropterins. S.W. Bailey, I. Rebrin, S.R. Boerth and J.E. Ayling. J. Am. Chem. Soc. 117, 10203-10211 (1995).

12. Activity of the Bifunctional Protein 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH during Human Fetal Development: Correlation with Dihydropteridine Reductase Activity and Tetrahydrobiopterin Levels. I. Rebrin, S.W. Bailey and J.E. Ayling. Biochem. Biophys. Res. Comm. 217, 958-965 (1995).

13. Total Chemical Synthesis of Chirally Pure (6S)-Tetrahydrofolic Acid. S.W. Bailey and J.E. Ayling. Methods in Enzymology 281, 3-16 (1997).

14. Mechanism of Dehydration by the Bifunctional Protein, 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH. J. E. Ayling, I. Rebrin, B. Thöny, and S. W. Bailey. Chemistry and Biology of Pteridines and Folates (Proceedings of the 11th International Symposium). Eds. W. Pfleiderer and H. Rokos. Blackwell Science, Berlin pp. 565-570 (1997).

15. Mechanism of Oxygen Activation by Phenylalanine Hydroxylase. M. D. Ardell, S. W. Bailey, and J. E. Ayling. Chemistry and Biology of Pteridines and Folates (Proceedings of the 11th International Symposium). Eds. W. Pfleiderer and H. Rokos. Blackwell Science, Berlin pp. 491-496 (1997).

16. Mechanistic Studies of 4a-Hydroxytetrahydropterin Dehydratase from Pseudomonas aeruginosa. I. Rebrin, J. Song, R. A. Jensen, and J. E. Ayling. Chemistry and Biology of Pteridines and Folates (Proceedings of the 11th International Symposium). Eds. W. Pfleiderer and H. Rokos. Blackwell Science, Berlin pp. 631-634 (1997).

17. Mutations in the Pterin-4a-Carbinolamine Dehydratase Gene (PCBD) are Causative for a Benign Form of Hyperphenylalaninemia. B. Thöny, F. Neuheiser, L. Kierat, M.O. Rolland, P. Guibaud, T. Schlüter, R. Germann, R.A. Heidenreich, M. Duran, J.B.C. de Klerk, J.E. Ayling, and N. Blau. Human Genetics 103, 162-167 (1998).

18. Hyperphenylalaninemia with High Levels of 7-Biopterin is associated with Mutations in the PCBD Gene Encoding the Bifunctional Protein Pterin-4a-Carbinolamine Dehydratase and Transcriptional Coactivator (DCoH). B. Thöny, F. Neuheiser, L. Keirat, M. Blaskovics, P.H. Arn, P. Ferreira, I. Rebrin, J.E. Ayling, and N. Blau. American Journal of Human Genetics 62, 1302-1311 (1998).

19. Stereospecificity and Catalytic Function of Histidine Residues in 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH. I. Rebrin, B. Thöny, S.W. Bailey, and J.E. Ayling. Biochemistry 37, 11246-11254 (1998).

20. Assessment of the effects of polymorphisms from analysis of intestine biopsies: Hyperphenylalaninemia associated with mutations in 4a-hydroxy-tetrahydropterin dehydratase. N. Blau, C.P. Braegger, R. Giugliani, S.R. Boerth, S.W. Bailey and J.E. Ayling. (1999).

21. J.E. Ayling, S.W. Bailey, S.R. Boerth, R. Giugliani, C. Braegger, B. Thöny and N. Blau. Hyperphenylalaninemia and 7-Pterin Excretion Associated with Mutations in 4a-Hydroxy-Tetrahydrobiopterin Dehydratase/DCoH: Analysis of Enzyme Activity in Intestinal Biopsies. Molecular Genetics and Metabolism 70: 179-188 (2000).

22. S.W. Bailey and J.E. Ayling. Food and Vitamin Preparations Containing the Natural Isomer of Reduced Folates. US Patent 6,254,904 (2001).

23. S.W. Bailey and J.E. Ayling. Compositions for Human and Animal Consumption Containing Reduced Folates and Methods for Making and Using Same. US Patent 5,997,915 (2000).

24. J.E. Ayling. The Uniquely Human Inefficient Conversion of Folic Acid to Tetrahydrofolates. Third Homocysteinemia and Atherosclerosis RFA Grantee’s Meeting, NIH, Bethesda, MD (2001).

25. J.E. Ayling. Stereospecific Synthesis of 2-Desamino-tetrahydropterins as Probes of Hydroxylase Cofactor Recognition. 12th Int. Symp. on Chemistry and Biology of Pteridines and Folates, Washington, DC (2001).

26. S.W. Bailey and J.E. Ayling. Method for Treating a Subject Afflicted with Intestinal Malabsorption. US Patent 6,451,360 (2002).

27. S. Vasudevan, S.W. Bailey, and J.E. Ayling. Stereospecific Synthesis of 2-Desamino-tetrahydropterins as Probes of Hydroxylase Cofactor Recognition. IN: S. Milstien (Ed.) Proceedings of the 12th International Symposium on Chemistry and Biology of Pteridines and Folates. Kluwer Publishers, Norwell, MA 37-41 (2002).