Crystal Structure of Rat GTP Cyclohydrolase I Feedback Regulatory Protein, GFRP Gerd Bader 1 , Susanne Schiffmann 2 , Anja Herrmann 2 , Markus Fischer 2 Markus Gu È tlich 2 , Gu È nter Auerbach 1 , Tarmo Ploom 1 , Adelbert Bacher 2 * Robert Huber 1 and Thorsten Lemm 1 1 Abteilung Strukturforschung Max-Planck-Institut fu Èr Biochemie, Am Klopferspitz 18a, D-82152 Martinsried Germany 2 Lehrstuhl fu È r Organische Chemie und Biochemie Technische Universita Èt Mu È nchen, Lichtenbergstr. 4 D-85747 Garching, Germany Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory pro- tein (GFRP). The 2.6 A Ê resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the inter- action of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins. # 2001 Academic Press Keywords: GTP cyclohydrolase I; tetrahydrobiopterin; biosynthesis; regulation; X-ray structure *Corresponding author Introduction GTP cyclohydrolase I (E.C. 3.5.4.16) catalyses the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate (H 2 NTP), the initial and rate limiting step in the de novo synthesis of tetrahydrobiopterin (BH 4 ). BH 4 is an essential cofactor for phenylalanine hydroxylase, tyrosine hydroxylase and tryptophane hydroxylase 1 and can affect the rate of biosynthesis of neurotransmit- ters such as serotonin or dopamine and hormones like melatonin via its intracellular concentration. Furthermore, BH 4 serves as a cofactor in all three forms of nitric oxide synthase 2 and glycerol ether monooxygenase. 3 As BH 4 , unlike other cofactors, is one of the regulators of these enzymic reactions, 4 it plays an important role in the control of phenyl- alanine catabolism and of neural and immune functions. Disorders in BH 4 synthesis are associ- ated with serious neuronal diseases such as hyperphenylalaninemia. 5 The intracellular BH 4 concentration is controlled by the rate of its de novo synthesis 1 by GTP cyclohydrolase I through transcriptional 6 and substrate 7 level regulators. Mammalian GTP cyclohydrolase I is subject to feedback inhibition by BH 4 mediated by complex formation with GTP cyclohydrolase I feedback regulatory protein (GFRP). 8 In the presence of GFRP and GTP, BH 4 inhibits GTP cyclohydrolase I while phenylalanine stimulates the enzyme. 9 This mode of GTP cyclohydrolase I regulation provides a mechanism to regulate phenylalanine catabolism and to avoid toxic accumulation of phenylalanine. BH 4 synthesis is also inhibited by various other pterins. The prototypic inhibitor 2,4-diamino-6- hydroxypyrimidine (DAHP) has been shown to act by a dual mechanism, dependent on, and indepen- dent of GFRP. 4 However, recent ®ndings indicate, that the regulation of GTP cyclohydrolase I activity might be different in different cell types as no GFRP mRNA was detectable in dopaminergic neu- rons. 10 It has also been shown that mammalian GTP cyclohydrolase I gets phosphorylated in vivo, 11 and that phoshorylation modulates GTP cyclo- hydrolase I activity. 12 GFRP has been puri®ed, sequenced and cloned from rat liver. 9 Crosslinking and sedimentation experiments indicate that the 9.5 kDa protein forms a pentamer. 13 Gel ®ltration and enzyme activity measurements indicate that in Present addresses: G. Auerbach, Antisense Pharma GmbH, Josef-Engert-Str. 9, D-93053 Regensburg, Germany; A. Herrmann, Microcoat GmbH, Am Neuland 1, D-82347 Bernried, Germany; T. Lemm, Roche Diagnostics, D-82327 Tutzing, Germany. Abbreviations used: BH 4 , tetrahydrobiopterin; H 2 NTP, dihydroneoptein triphosphate; GFRP, GTP cyclohydrolase I feedback regulatory protein; DAHP, 2,4-diamino-6-hydroxypyrimidine; CBP, calmodulin- binding peptide. E-mail address of the corresponding author: adelbert.bacher@ch.tum.de doi:10.1006/jmbi.2001.5011 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 312, 1051±1057 0022-2836/01/051051±7 $35.00/0 # 2001 Academic Press