Pterin reductase

FIGURE 18.35 Formation of THF from folic acid by the dihydrofolate reductase reaction. The R group on these folate molecules symbolizes the one to seven (or more) glutamate units that folates characteristically contain. All of these glutamates are bound in y-carboxyl amide linkages (as in the folic acid structure shown in the box A Deeper Look Folic Acid, Pterins, and Insect VFingis). The one-carbon units carried by THF are bound at N, or at or as a single carbon attached to both... 

The molyhdopterin cofactor, as found in different enzymes, may be present either as the nucleoside monophosphate or in the dinucleotide form. In some cases the molybdenum atom binds one single cofactor molecule, while in others, two pterin cofactors coordinate the metal. Molyhdopterin cytosine dinucleotide (MCD) is found in AORs from sulfate reducers, and molyhdopterin adenine dinucleotide and molyb-dopterin hypoxanthine dinucleotide were reported for other enzymes (205). The first structural evidence for binding of the dithiolene group of the pterin tricyclic system to molybdenum was shown for the AOR from Pyrococcus furiosus and D. gigas (199). In the latter, one molyb-dopterin cytosine dinucleotide (MCD) is used for molybdenum ligation. Two molecules of MGD are present in the formate dehydrogenase and nitrate reductase. 

The molybdenum cofactor was liberated from D. gigas AOR, and under appropriate conditions was transferred quantitatively to nitrate reductase in extracts of Neurospora crassa nit-1 mutant) to yield active nitrate reductase 217). On the basis of molybdenum content, the activity observed for reconstitution with molybdenum cofactor of D. gigas was lower (25%) than the values observed for the procedure using extractable molybdenum cofactor of XO, used as reference. This result can now be put in the context of the difference in pterin present (MPT-XO and MCD-AOR) 218). 

D. desulfuricans is able to grow on nitrate, inducing two enzymes that responsible for the steps of conversion of nitrate to nitrite (nitrate reductase-NAP), which is an iron-sulfur Mo-containing enzyme, and that for conversion of nitrite to ammonia (nitrite reduc-tase-NIR), which is a heme-containing enzyme. Nitrate reductase from D. desulfuricans is the only characterized enzyme isolated from a sulfate reducer that has this function. The enzyme is a monomer of 74 kDa and contains two MGD bound to a molybdenum and one [4Fe-4S] center (228, 229) in a single polypeptide chain of 753 amino acids. FXAFS data on the native nitrate reductase show that besides the two pterins coordinated to the molybdenum, there is a cysteine and a nonsulfur ligand, probably a Mo-OH (G. N. George, personal communication). 

R. L. Blakley, Dihydrofolate reductase, in Folates and Pterins, Chemistry and... 

Fe 2S], a [4Fe-4S] and a [3Fe-4S] center. The enzyme catalyzes the reversible redox conversion of succinate to fumarate. Voltammetry of the enzyme on PGE electrodes in the presence of fumarate shows a catalytic wave for the reduction of fumarate to succinate (much more current than could be accounted for by the stoichiometric reduction of the protein active sites). Typical catalytic waves have a sigmoidal shape at a rotating disk electrode, but in the case of succinate dehydrogenase the catalytic wave shows a definite peak. This window of optimal potential for electrocatalysis seems to be a consequence of having multiple redox sites within the enzyme. Similar results were obtained with DMSO reductase, which contains a Mo-bis(pterin) active site and four [4Fe 4S] centers. 

Figure 17.2 The structure of the pterin cofactor (1) which is common to most molybdenum- and tungsten-containing enzymes and schematic active site structures for members of the xanthine oxidase (2,3), sulfite oxidase (4) and DMSO reductase (5-7) enzyme families. (From Enemark et al., 2004. Copyright (2004) American Chemical Society.)...

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

The expansion in the power of computers and theoretical methods has made it possible to investigate the mechanism of action of enzymes by combinations of quantum-mechanical and molecular-mechanical calculations. A study of two possible mechanisms for dihydrofolate reductase catalysis was consistent with indirect proton transfer from aspartate to N-5 of the pterin as has been suggested for many years by crystallographic evidence <2003PCB14036>. This conclusion is also consistent with the outcome of a study that directly measured the of the active site aspartate in the Lactobacillus casei enzyme <1999B8038>. Observations of chemical shifts of... 

BH4 is essential for the AAHs to carry out their respective catalytic reactions and, at least for PAH, the prereductive activation, which appears to produce dihydrobiopterin quinonoid (g-BH2) directly (20). After the PAH catalytic cycle an oxygen atom is incorporated into the cofactor, providing 4a-OH-BH4 which dissociates from the active site. In order to regenerate the functional tetrahydro form of BH4 pterin carbinolamine dehydratase catalyzes the dehydration of 4-OH-BH4 to g-BH2, which is reduced back to by dihydropteridine reductase (Scheme 2). g-BH2 can also be converted to 7,8-dihydropterin (BH2) which can be regenerated to BH4 by dihydrofolate reductase (DHFR). 

A Fig. 6.1.7a- HPLC of pterins using a column-switching system a standard mixture b control urine c urine guanosine triphosphate cyclohydrolase I (GTPCH) deficiency d urine 6-pyru-voyl-tetrahydropterin synthase (PTPS) deficiency e urine pterin-4a-carbinolamine dehydratase (PCD) deficiency f urine dihydropteridine reductase (DHPR) deficiency g urine phenylketonuria 4-8 h after tetrahydrobiopterin (BH4) administration h-k see next page... 

Fig. 6.1.8a-c HPLC of the yellow-fluorescing pterins, a Standard mixture b control cerebrospinal fluid (CSF) c CSF sepiapterin reductase deficiency. HS -Hydroxyse-piapterin, S sepiapterin, X xanthopterin... 

DHPR Dihydropteridine reductase, DRD dopa-responsive dystonia, GTPCH GTP cyclohydrolase I, n normal, PCD pterin-4a-carbinolamine dehydratase, PTPS 6-pyruvoyltetrahydrobiopterin synthase, SR sepiapterin... 

The synthesis of functionalized 7,7-dialkyl-7,8-dihydropterins and a 7-spirocyclopropane derivative have been performed to find new types of inhibitors for 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and dihydrofolate reductase <85JCS(Pl)2133, 85JCS(P1)2145, 89JCS(P1)1297>. An informative review on the chemical and biological reactions of reduced pterins is also available <84MI 718-08). 

Figure 2. The ligand common to all molybdenum and tungsten enzymes, MPT, is shown here in several formats (a) in common stick notation (b) as a ball and stick (c) an orientation rotated 90° from view (b) to emphasize the spacial relationship between the pterin plane and the dithiolene-pyran ring portion (d) MGD in common stick notation and for comparison, (e ) FAD, a common electron-transfer prosthetic group. Coordinates for the views in (b) and (c) are taken from the data deposited in the Protein Data Bank (PDB) for the 1.3-A resolution structure of DMSO reductase from Rhodobacter sphaeroides.

---