JBC Papers in Press. Published on April 12, 2011 as Manuscript M111.219576 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M111.219576 QUEUOSINE DEFICIENCY IN EUKARYOTES COMPROMISES TYROSINE PRODUCTION THROUGH INCREASED TETRAHYDROBIOPTERIN OXIDATION Tatsiana Rakovich‡, Coilin Boland‡, Ilana Bernstein§, Vimbai M. Chikwana¶, Dirk Iwata-Reuyl¶, and Vincent P. Kelly‡ From the ‡School of Biochemistry & Immunology, Trinity College Dublin, Dublin 2, Ireland, §School of Environmental & Life Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia, and ¶Department of Chemistry, Portland State University, Portland, Oregon 97207 Running head: Queuosine deficiency leads to elevated dihydrobiopterin Address correspondence to: Vincent Kelly, Ph.D., School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland. Tel: +353-1-8963507; Fax: 81-298-53-7318; E-mail: [email protected] Queuosine is a modified pyrrolopyrimidine of complex carbohydrates to the provision of vital nucleoside found in the anticodon loop of micronutrients (1). Queuosine is an example of a transfer RNA acceptors for the amino acids micronutrient, synthesised exclusively by bacteria Downloaded from tyrosine, asparagine, aspartic acid, and but which, for poorly defined reasons, is utilised histidine. Since it is exclusively synthesised by by almost all eukaryotic species with the exception bacteria, higher eukaryotes must salvage of the baker’s yeast, S. cerevisiae (2). queuosine or its nucleobase queuine from food Bacterial queuosine biosynthesis occurs in two and the gut microflora. Previously, animals stages. Firstly, a series of five enzymatic steps www.jbc.org made deficient in queuine died within 18 days of convert guanosine triphosphate nucleoside (GTP) withdrawing tyrosine—a non-essential amino to the soluble 7-aminomethyl-7-deazaguanine acid—from the diet [Marks T, Farkas WR (preQ ) molecule. Subsequently, preQ is inserted 1 1 at IReL (Trinity College Dublin), on May 23, 2011 (1997) Biochem Biophys Res Commun 230:233- into the wobble position of tRNA containing a 7]. Here we show that human HepG2 cells GUN consensus sequence (tyr, asp, asn, his) by deficient in queuine and mice made deficient in means of the single enzyme species, tRNA guanine queuosine modified transfer RNA, by transglycosylase (TGT) and further remodeled in disruption of the tRNA guanine transglycosylae situ to queuosine (3). Eukaryotes must acquire (TGT) enzyme, are compromised in their ability queuosine or its free nucleobase, queuine, from to produce tyrosine from phenylalanine. This food and the gut-microflora. Curiously, both has similarities to the disease phenylketonuria, cytosolic and mitochondrial tRNA species are which arises from mutation in the enzyme modified by queuosine (2). The eukaryotic enzyme phenylalanine hydroxylase or from a decrease that performs this reaction, queuine tRNA in the supply of its cofactor tetrahydrobiopterin ribosyltransferase, has recently been identified as a (BH4). Immunoblot and kinetic analysis of liver heterodimeric complex, consisting of the from TGT deficient animals indicate normal eukaryotic homologue of the catalytic TGT subunit expression and activity of phenylalanine and a related protein called queuine tRNA hydroxylase. By contrast, BH4 levels are ribosyltransferase domain containing 1 (QTRTD1), significantly decreased in the plasma and both both of which localize to the mitochondria (4, 5). plasma and urine show a clear elevation in Studies on germ-free (axenic) mice maintained dihydrobiopterin, an oxidation product of BH4, on a chemically defined diet, provided clear despite normal activity of the salvage enzyme evidence that eukaryotes are non-autotrophic for dihydrofolate reductase. Our data suggest that queuosine biosynthesis (6). Unchallenged, these queuosine modification limits BH4 oxidation in animals appear normal. However, withdrawal of vivo and thereby potentially impacts on tyrosine from the diet resulted in symptoms of numerous physiological processes in eukaryotes. squinting, stiffness, lethargy, convulsion, and ultimately death after 18 days (7). Re- Bacteria and humans have co-evolved for millenia administration of either chemically synthesised and many examples exist of how various symbiotic queuine or tyrosine alone prevented the symptoms; and commensal partnerships contribute to human the latter result suggesting that tyrosine uptake and health and nutrition ranging from the metabolism utilisation is unaffected by queuine status. It has 1 Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc. been long established that tyrosine is a non- Animals: Mice were bred and housed under essential amino acid in higher eukaryotes as it can specific pathogen-free conditions. Procedures were be synthesized from phenylalanine by the action of performed on mice at 6–8 weeks of age, unless the phenylalanine hydroxylase (PAH) enzyme. It otherwise stated, according to regulations and has been suggested therefore that the absence of guidelines of the Ethics Committee, Trinity queuine may affect the translation of the PAH College Dublin and the Irish Department of Health. enzyme leading to a dietary dependency on Tyrosine free chemically defined diet (AIN-76A tyrosine supply (2, 8). purified diet) was obtained from Harlan Teklad. In humans, tyrosine production occurs principally in the liver and kidney correlating with Cell Culture: HepG2 cells were purchased from the expression of the PAH enzyme (9). Deficiency the American Tissue Culture Collection and grown in PAH leads to the disease phenylketonuria, in an atmosphere of 5% CO2 at 37°C in the characterized by increased blood levels of following media: Dulbecco's modified Eagle's phenylalanine (referred to hyperphenylalaninemia) medium (DMEM) supplemented with 10% (v/v) and reduced levels of tyrosine. In performing its fetal bovine serum (DMEM-FBS) or 10% (v/v) Downloaded from reaction PAH requires molecular oxygen and BH4 horse serum (DMEM-HS), 2 mM L-glutamine, 1 cofactor (Fig. 1). BH4 is produced from GTP by mM sodium pyruvate, and penicillin/streptomycin the enzymes GTP cyclohydrolase I, 6-pyruvoyl- (50 U/0.1 mg/ml) or in Ultraculture™ serum free tetrahydropterin synthase and sepiapterin reductase. medium (Lonza) supplemented with 2 mM L- In generating BH4, other intermediary reactions at glutamine and penicillin/streptomycin (10 U/20 www.jbc.org the sepiapterin reductase step are performed by µg/ml). When required, chemically synthesized carbonyl reductase and member proteins of the queuine (a gift from Dr. Susumu Nishimura, aldo-keto reductase family (10). The BH4 cofactor Tsukuba University, Japan) was added at a final at IReL (Trinity College Dublin), on May 23, 2011 may also be recycled by the activity of two concentration of 300 nM. enzymes, pterin-4a-carbinolamine dehydratase and dihydropteridine reductase, which has particular Radiolabelled phenylalanine hydroxylase assay: importance for tyrosine biosynthesis in the liver. HepG2 cells were grown in serum-free (SF) Deficiency in any of the BH4 enzymes, with the medium, SF medium supplemented with queuine exception of sepiapterin reductase, causes (300nM) or in DMEM with 10% FBS. Intracellular hyperphenylalaninemia. Recent studies have tyrosine and phenylalanine were depleted by shown that BH4 is also highly susceptible to auto- incubation in SF medium not containing tyrosine oxidation in vivo producing the metabolite 7,8 or phenylalanine (Lonza; custom synthesis) and dihydrobiopterin (BH2), whose accumulation is supplemented with cyclohexamide (20µg/ml) with limited by the enzyme dihydrofolate reductase constant shaking at 70 rpm for 1 hour. (DHFR) through reduction of BH2 to BH4 (11). Subsequently, cells were washed and incubated in By extrapolation from the current data, the KRHL buffer (120 mM NaCl, 4.8 mM KCl, 10 tyrosine dependency of queuine deficient animals mM D-glucose, 2.5 mM CaCl2, 1.3 mM MgSO4, may relate to the loss of PAH activity or altered 2.5 mM Hepes, pH 7) containing 20 µM, 60 µM BH4 cofactor supply. In the present study, we and 100 µM L-[2,6-3H]- Phenylalanine (54 show that queuosine modification of tRNA, or Ci/mmol; Amersham) and 20 µg/ml hypothetically another unknown RNA substrate of cyclohexamide for 30 minutes with constant the queuine tRNA ribosyltranferase enzyme, as shaking. Duplicate wells for each experimental opposed to free queuine base, is required for point were used to determine cell protein content. normal tyrosine production in eukaryotes. Cells were solubilised with M-PER solution Decreased BH4 levels, concomitant with a marked (Pierce) and the protein concentration determined accumulation of BH2 suggests that oxidation of by Bradford assay (Bio-Rad). After 30 minutes, BH4 cofactor underlies the defect. medium was removed, cells were washed rapidly with ice-cold PBS and lysed in ice-cold 10% EXPERIMENTAL METHODS trichloroacetic acid (100 µl per well). Cells were scraped from the dish and the lysate centrifuged at 16,000 x g for 15 minutes. The supernatant was 2 stored at -70 oC until analysed. Intracellular PAH cDNA was cloned into the BamHI and SalI phenyalanine and tyrosine were measured by restriction sites of pMal-c2 (New England Biolabs) HPLC on a Zorbax 300SB-C18 column (Agilent) which was transformed in E.coli BL21 DE3 cells pre-equilibrated with Mobile Phase Buffer (100 Protein induction was performed by addition of 0.5 mM sodium phosphate pH 1.9, 300 µM octyl mM isopropyl-1-thio-β-D-galacto-pyranoside at sodium