Tyrosinemia I, A Model For Human Diseases Mediated By 2-Oxoacid-Utilizing Dioxygenases: Hepatotoxin Suppression By NTBC Does Not Normalize Hepatic Collagen Metabolism *Hartmut M. Hanauske-Abel, ‡Anthony Popowicz, †Helen Remotti, *Ron S. Newfield, and *Joseph Levy Departments of *Pediatrics and †Pathology, New York-Presbyterian Hospital, Columbia and Cornell Campuses, and ‡Computing Services, Rockefeller University, New York, New York, U.S.A. ABSTRACT Objectives: Medical treatment of tyrosinemia I relies on the herbicide NTBC [Orfadin; 2-(2-nitro-4-trifluoromethylbenzoyl)- cyclohexane-1,3-dione], an inhibitor of plant and mammalian 2-oxoacid-utilizing dioxygenases with a collective catalytic cycle (‘HAG’ mechanism). We hypothesize that NTBC-treated tyrosinemia I is a human model for the pathogenic role of two major enzymes in this class, 4-hydroxyphenylpyruvate dioxy- genase (4-HPPD; EC 1.13.11.27) and prolyl 4-hydroxylase (P4-H; E.C. 1.14.11.2), essential for tyrosine and collagen me- tabolism, respectively. Methods: In a patient with established tyrosinemia I, we moni- tored the in vivo activities of 4-HPPD and P4-H via five bio- markers before and during NTBC medication. Hypothesis test- ing at the molecular level was performed by computational modeling of NTBC binding to the crystal structure-derived ac- tive site of 4-HPPD, and then relating these findings to our experimental results and to known P4-H data. Results: NTBC rapidly normalized the biomarkers for 4-HPPD activity. However, those for P4-H activity remained uniformly elevated after one hundred days on NTBC, the PIIINP biomar- ker even increasing above its grossly abnormal, initial level. This selective enzyme inhibition despite a collective catalytic cycle is attributed to the conformation of NTBC, which only fits the active site of 4-HPPD, as confirmed by its crystal struc- ture. Conclusions: Normalization of hepatic collagen formation, highly desirable in all fibrotic liver diseases, is not achieved by NTBC in tyrosinemia I. By establishing the molecular cause for this failure, our results also establish a rational approach to identify inhibitors that achieve that goal, either by joint 4-HPPD / P-4H inhibition, or by inhibition of only P-4H. JPGN 35:73–78, 2002. Key Words: Tyrosinemias— Cyclohexanones—4-hydroxyphenylpyruvate dioxygenase— Procollagen-proline dioxygenase— Drug design. © 2002 Lip- pincott Williams & Wilkins, Inc. INTRODUCTION Tyrosinemia I, a recessively inherited disease that pro- foundly disrupts liver and kidney function, is caused by the deficiency of fumarylacetoacetate hydrolase (EC 3.7.1.2). The pathogenesis of potentially lethal liver damage in tyrosinemia I involves, however, two addi- tional enzymes: 4-hydroxyphenylpyruvate dioxygenase (4-HPPD; EC 1.13.11.27), which forms the actual hepa- totoxins; and prolyl 4-hydroxylase (P-4H; E.C. 1.14.11.2), required for the reactive hepatic fibrosis and indispensable for collagen synthesis in general. 4-HPPD and P4-H are non-heme iron enzymes, and each con- sumes one molecule of atmospheric oxygen and one 2-oxoacid moiety while hydroxylating one carbon atom. 4-HPPD and P-4H have long been classified together as 2-oxoacid-utilizing dioxygenases (1), and therefore tyro- sinemia I ranks as a model for human diseases mediated by this class of enzymes, diseases that prominently in- clude all fibrotic conditions. A single, collective catalytic pathway—the ‘HAG’ mechanism, proposed in 1982 by Hanauske-Abel and Günzler—resolves the substrate alignment, catalytic orbital interactions, and product re- lease of the 2-oxoacid-utilizing dioxygenases (2–4), and has been extremely useful for the discovery of inhibitors and candidate drugs targeting these enzymes (5,6). As expected, the HAG mechanism-utilizing enzymes dis- play a remarkable cross-species susceptibility to such inhibitors; e.g., human and plant 4-HPPD are similarly Received May 24, 2001; accepted October 23, 2001. Address correspondence and requests for reprints to Hartmut M. Hanauske-Abel, M.D., Ph.D., Evariste Galois Foundation, 1055 River Road, PO Box 66, Edgewater, NJ 07020, U.S.A. (e-mail: hmhanaus@ earthlink.net). Extramural grant support: National Institutes of Health (RR06020 and HD00072) Journal of Pediatric Gastroenterology and Nutrition 35:73–78 © July 2002 Lippincott Williams & Wilkins, Inc., Philadelphia 73 DOI: 10.1097/01.MPG.0000017326.32621.7D