John M. Whiteley et uf. : Dihydropteridine reductase Pteridines Vol. 4, 1993, pp. 159-173 Review Dihydropteridine Reductase John M. Whiteley§, Kottayil I. Varughesej:, Nguyen H. Xuong#, David A. Matthews, t and Charles E. Grimshawf §The Scripps Research Institute, La Jolla, CA 92037, USA., #University of California at San Diego, La Jolla, CA 92093-0317, U.SA., tAgouron Pharmaceuticals, Inc., San Diego, CA 92121, USA, and fThe Whittier Institute, La Jolla, CA 92037, USA. (Received August lO, 1993) Summary During the past decade numerous advances have been made in understanding the structure, mechanism and clinical properties of dihydropteridine reductase. An attempt is made here to delineate the current status of this essential enzyme by describing its structural features, its kinetic mechanism, the cloning and expression of both rat and human enzyme forms, the solution of their crystal structures, their classifica­ tion as members of a large family of short chain dehydrogenases, and finally a brief description is included indicating how current molecular biological applications have allowed the clinical definition of the aberrant form of phenylketonuria caused by a defective reductase. Key words: Dihydropteridine reductase, Quinonoid dihydrobiopterin, Crystal structure, Aberrant PKU, Gene expression, Mutagenesis Introduction and history in their heterocyclic nucleus of many centers for protonation, which often influence binding and reac­ Naturally occurring pteridines, which usually con­ tivity. Important metabolic functions of conjugated tain 2-amino and 4-hydroxyl substituents can be sep­ pteridine-mediated biological reactions include the arated into two distinct classes. One class contains one-carbon insertion reactions fundamental to pu­ the pterins of the folic acid series which possess rine biosynthesis (4. 5), the vitamin B,Tmediated syn­ a p-aminobenzoylglutamate (or polyglutamate) moie­ thesis of methionine (6), and the synthesis of thymi­ ty substituted into the heterocyclic nucleus via a 6- dylate (7). The most important unconjugated pteri­ methylene group, and the second class contains mo­ dine-containing enzymatic systems are associated lecules possessing other substituents at the 6-position, with the metabolism of the aromatic amino acids e.g., biopterin, neopterin and xanthopterin. Since (8-10). the discovery and classification of pteridines in Dihydropteridine reductase and phenylalanine. ty­ the early part of this century (1 , 2) a large number rosine and tryptophan hydroxylases play vital roles of pteridine-or folate-requiring enzymes have been in the synthesis of the catecholamines dopamine. identified and their mechanisms of action examined epinephrine and serotonin and indirectly can also (3). Many investigators have been intrigued by the intluence the generation of the melanin pigments. diverse functions of pteridines when they act as co­ Tetrahydrobiopterin is an essential cofactor in these factors or substrates in enzymatic reactions, by their metabolic pathways and facilitates the monooxygen­ unusual organo-chemical properties. by their pref­ ase activity which ultimately leads to the hydroxyl­ erence for hydroxylic solvents and by the presence ation of the aromatic amino acid substrate (Figure Plcridines / Vol.4 / No. 4 160 John M. Whiteley el al.: Dihydropteridine reductase protocol ineffective treatment for this group of pa­ tients and emphasizes the importance of characterising Phenylalanine Tryptophan ~~ NAO" these alternate defects. Currently, diagnosis is usually Tyrosine ~: achieved by further serological analyses (17), but H,N H H clearly a more precise understan·ding of DHPR ac­ T etrahydro bi opterln Aromatic tion could be of value in interpreting clinical situa­ Amino Acid Hydroxylases tions relating to a malfunctioning reductase. For example, if the genetic defect in DHPR deficient + H 0 2 patients can be identified, as has in fact been recent­ Tyrosine 01 hyd roxy p h e ny lal ani ne ly reported for several patients (18, 19), the lesion 5-hyd roxytryptophan H,NLa: H H can be related to the structure, and the cause for Hqulnonoid" dihydrobiopterin diminished enzymatic activity may be identified at Figure I. The participation of DHPR in the conversion of the molecular level. In addition, as the data base quinonoid dihydrobiopterin (R=dihydroxypropyl) to tetra­ of defective structures increases, a possible pattern hydrobiopterin, which is used as a cofactor in the aromatic of common causal features may evolve via correla­ amino acid hydroxylations. tion with the X-ray crystallographic s~ructure and targets may he created for molecular corrective ac­ 1). In these reactions phenylalanine, tyrosine and tion. tryptophan are converted to tyrosine, dihydroxyphen­ The purpose of this review article is to outline ylalanine and 5-hydroxytryptophan, and the cofac­ current knowledge relating to DHPR with a particu­ tor undergoes oxidation to a quinonoid dihydro lar emphasis on more recent developments in the structural and mechanistic understanding of this en­ form of biopterin (q-BH2). q-BH2 then becomes the substrate for DHPR and in an NADH-mediated zyme. Excellent reviews by Annarego et al. (20) and Shiman (8) have covered many of the salient fea­ reaction is recycled to BH4. The principal metabolic end products of these BH4-dependent amino acid tures of this enzyme and therefore areas covered in hydroxylations have important cellular functions. depth by these earlier reviews will receive only limit­ Dopamine (3,4-dihydroxyphenyIethylamine) is a cen­ ed coverage in the current report. tral nervous system neurotransmitter representing more than 50% of the total catecholamine content in the brain and spinal cord of many species (II). Source, purification and properties of DHPR and norepinephrine (3,4-dihydroxyphenylethanol­ amine) is the chemical transmitter in sympathetic DHPR has been obtained from several mamma­ neurons and is an essential neurotransmitter in lian sources that include sheep brain and beef adre­ brain tissue. Epinephrine (N-methyl-l(3-4-dihydroxy­ nal medulla (21), the liver of sheep (22), beef (23). phenyl)ethanolamine) is best known as an important rat (24, 25), monkey and human (26, 27), and hormone of the peripheral atKonomic system, al­ also from various murine (28) and human cell sam­ though it also functions as a transmitter in the olfac­ ples (29, 30). In addition, the enzyme has been isolat­ tory system and as a central nervous system neuro­ ed from species of Pseudomonas (31), E~cherichia coli transmitter (12). Serotonin (5-hydroxytryptamine) is (32), and Crithidia fasciculata (33). From mammalian an important brain neurotransmitter that exerts wi­ sources the tissue is usually diced and homogenised despread influence over arousal, sensory perception, in the presence of buffer or dilute acetic acid contain­ emotion and higher cognitive functions (13). ing a cocktail of protease inhibitors and solid ma­ The defective function of the hydroxylation pro­ terial is separated by centrifugation. The active com­ cess has long been recognized clinically in the auto­ ponent is then concentrated by ammonium sulfate somal recessive disease hyperphenylalaninemia or fractionation and after dialysis is subjected to a va­ phenylketonuria (PKU) (14). Originally this disease riety of chromatographic separation procedures. The was considered to correlate only with a defective earlier methods relied heavily on DEAE-cellulose phenylalanine hydroxylase, but a significant number or-sephadex fractionation but more recent efforts of cases have now been identified whose cause lies have employed specific affinity matrices or dye con­ in a defective DHPR function or aberrant biosyn­ taining matrices such as Procion Red or Cibacron thesis of the tetrahydrobiopterin cofactor (15,16). The Blue. In some instances a further hydrophobic chro­ enhanced complexity resulting from an additional matographic step followed by Sephadex or Sepha­ requirement for the cofactor in catecholamine biosyn­ cryl sizing columns has allowed isolation of a com­ thesis makes the standard phenylalanine-free diet pletely homogeneous product (34). A typical protocol P :~ri di llcS / Vol.4 / NO.4 John M. Whiteley er al.: Dihydropteridine reductase 161 Table I . The Purification of the E. coli expressed human DHPR Volume Enzy me Protein Specific Activity Recovery Purification Step (ml) (units) (mg) (units/ mg) (%) - - - - --- ----------------------------- - -----,,-,,-- Acetic Acid Extract 310 4200 (N H4)2S0.(21-45%) 112 1900 1st Cibacron Blue 282 21.630 725 29.8 100 2nd Cibacron Blue 186 19,400 69 281 90 Sephadex G-150 72 16,900 41 412 78 is shown in Table 1. It is interesting to note the progression of enhanced specific activities (from 2.5- -:>400 units/mg protein) over the years with the improvement in purification procedures. It is evident that the blue dye column materials, known to recog­ .2m(b) nise dinucleotide protein binding sites, are particu­ larly effective in ensuring a product of higher specific Figure 2. The comparative structures of the quinonoid dihy­ activity is obtained (35). This observation correlates dropteridine (a) and the 7,8-dihydropteridine (b). well with the structural and kinetic properties of the enzymes, outlined elsewhere in this report, that illustrate the enhance stability of the protein when (ku-O.02 f,lM) over NADPH (kct-2.2 f,lM), although bound to its dinucleotide cofactors. The isolation the possibility does exist that in vivo the relative con­ of the bacterial and flagellate reductases