Dihydropyrimidinases

DIHYDROOROTATE DEHYDROGENASE DIHYDROOROTATE OXIDASE DIHYDROPTERIDINE REDUCTASE DIHYDROPTEROATE SYNTHASE DIHYDROPYRIMIDINASE DIHYDROURACIL DEHYDROGENASE Dihydroxyacetone kinase,... 

Kuhara T, Ohdoi C, Ohse M et al. Rapid gas chromatographic-mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency. J Chromatogr B Analyt Technol Biomed Life Sci 2003 792 107-115. 

Fig. 7.1.3 High-performance liquid chromatography/electron spin ionization-tandem mass spectrometry profiles of urines from patients with a dihydropyrimidine dehydrogenase (upper panel), dihydropyrimidinase (middle panel) and a /Tureidopropionase deficiency (lower panel). The inserts show the profiles of a control urine, note the differences in scale. N-C-fi-ala-nine N-Carbamyl-j6-alanine, N-C-fi-AIB N-carbamyl-/Taminoisobutyric acid...

Table 7.1.4 Concentration range of purine and pyrimidine metabolites in urine (pmol/mmol creatinine) from patients. ADA Adenosine deaminase, APRT adenine phosphoribosyltransferase, ASA adenylosuccinate lyase, DHP dihydropyrimidinase, DPD dihydropyrimidine dehydrogenase, HGPRT hypoxanthine-guanine phosphoribosyltransferase, PNP purine nucleoside phosphorylase, TP thymidine phosphorylase, UMPS uridine monophosphate synthase, / -UP fi-ureidopropionase... 

Bacterial contamination of the urine may result in strongly increased levels of uracil due to the bacterial degradation of pseudouridine. Thymine-uraciluria, which is indicative of a dihydropyrimidine dehydrogenase or dihydropyrimidinase deficiency, may also result from increased tissue degradation. However, the latter situation is also characterized by hyper-/l-aminoisobutyric aciduria and hyper-/f-alaninuria [6]. Under alkaline conditions, due to the presence of bacterial contamination, the de-oxynucleosides may be hydrolyzed toward their corresponding nucleoside bases. 

The activity of dihydropyrimidinase or /J-urcidopropionasc can only be measured in liver or kidney. The activity of dihydropyrimidinase is determined using a radiochemical assay with subsequent separation of radiolabeled dihydrouracil from radiolabeled N-carbamyl-/>-alanine with reverse-phase HPLC combined with detection of 14C02by liquid scintillation counting [11]. The activity of /1-ureidopropionase can be determined using radiolabeled N-carbamyl-/l-alanine followed by separation of radiolabeled N-carbamyl-/>-alanine from radiolabeled /1-alanine by reverse-phase HPLC [10,14]. 

Van Kuilenburg ABP, van Lenthe H, van Kuilenburg ABP (1999) Radiochemical assay for determination of dihydropyrimidinase activity using reversed-phase high-performance liquid chromatography. J Chromatogr Biomed Sci Appl 729 307-314... 

The bacterial D-hydantoinase has been isolated as crystals from cells of Pseudomonas putida (= P. striata) (Table 1) [5]. Because the purified enzyme showed the highest activity and affinity toward dihydrouracil, the enzyme was identified as dihydropyrimidinase (EC. 3.5.2.2). Interestingly, the enzyme also attacked a variety of aliphatic and aromatic D-5-mono-substituted hydantoins, yielding the corresponding D-form of N-carbamoyl-a-amino acids. Thus, the enzyme can be used for the preparation of various D-amino acids. Under the conditions used for the enzymatic hydrolysis of hydantoin at pH 8 to 10, the L-isomers of the remaining hydantoins are racemized through base catalysis. Therefore, the racemic hydantoins can be converted quantitatively into N-carbamoyl-D-amino acids through this step. 

Many kinds of enzymes with different substrate specificities are involved in hydantoin hydrolysis. Ogawa et al. [10] found two hydantoin-hydrolyzing enzymes in Blastobacter sp. A17p-4. These enzymes were purified to homogeneity and characterized (Table 1). One hydrolyzed dihydropyrimidines and 5-monosubstituted hydantoins to the corresponding AT-carbamoyl amino acids. Since the hydrolysis of 5-substituted hydantoins by this enzyme was D-stereo-specific, this enzyme was identified as D-hydantoinase, which is identical with dihydropyrimidinase. The other one preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds such as dihydrouracil and hydantoin. Because there have been no reports on enzymes which show same substrate specificity as this enzyme, it is considered to be a novel enzyme, which should be called imidase [10]. 

Since D-hydantoinase was identified as dihydropyrimidinase, it is proposed that D-amino acid production from DL-5-monosubstituted hydantoins involves the action of the series of enzymes involved in the pyrimidine degradation pathway. Based on this proposal, D-decarbamoylase was thought to be identical with P-ureidopropionase (EC 3.5.1.6) which functions in pyrimidine metabolism. 

Hydantoinases and decarbamoylases have been applied for the production of optically active amino acids from DL-5-monosubstituted hydantoins. A variety of enzymes have been reported elsewhere. Runser et al. [33] reported the occurrence of D-hydantoinase without dihydropyrimidinase activity. Watabe et al. [34] reported that an ATP-dependent hydantoin-hydrolyzing enzyme is involved in the L-amino acid production from DL-5-monosubstituted hydantoin by Pseudomonas sp. NS671. This enzyme shows no stereospecificity. Hydan-toinase showing no stereospecificity and not requiring ATP was also reported [35]. Recently, hydantoin-racemizing enzymes were found [36,37], These enzymes make it possible to totally convert racemic substrates, which only slowly racemize under reaction conditions, to a single stereoisomer. The combinations of these hydantoin-transforming enzymes provide a variety of processes for optically active amino acid production (Fig. 4). 

D-Hydantoinase identical with dihydropyrimidinase Pseudomonas Blastobacler etc. 

Another example of screening was for the dihydropyrimidinase enzymes (E.C. 3.5.2.2), also known as hydantoinases, used for the production of either l- or D-amino acids.407,408 These studies... 

In the second step the dihydropyrimidines obtained are hydrolyzed by f-dihydropyrimidinase (EC 3.5.2.2) to N-carbamoyl-/i-alanine 6 and N-carbamoyl-/i-aminoisobutyrate 9, respectively (Scheme 1.6.2). The enzyme isolated from rat liver was purified, characterized [8] and cloned [9]. 

M., Booze, R., Markesbery, W. R., Butterfield, D. A. Proteomic identification of oxidatively modified proteins in Alzheimer s disease brain part II. Dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate. /. Neurochem. 2002,71 1524-1532. 

Two other genes have been linked to deficit patients dihydropyrimidinase-related protein 2 (DRP-2) and guanine nucleotide-binding protein (G-protein) alpha stimulating activity polypeptide 1 (GNAS1). 

Fountoulakis, M. (1999). Expression of the dihydropyrimidinase related protein 2 (DRP-2) in Down syndrome and Alzheimer s disease brain is down-regulated at the mRNA and dysregulated at the protein level. J. Neural. Transm. Suppl. 57,161-177. 

Peripheral mononuclear cells do not have dihydropyrimidinase, the second step in conversion of 5-FU to fluoro-P-alanine. Three peaks are not always seen, depending on the HPLC column and conditions. 

Dihydropyrimidinase, an enzyme responsible for the hydrolytic ring opening reactions of dihydrothymine (16) and dihydrouracil (17), was partially purified and characterized from calf and rat liver. The enzyme from both sources catalyzed the hydrolytic ring-opening of dihydrouracil, hydantoin (18), 5-phenylhydantoin (19), and a-phenylsuccinimide (20). 

In mammalian systems, catabolism of uracil and thymine proceeds in parallel steps, catalyzed by the same enzymes (Figure 27-31). The rate-determining step is reduction to a 5,6-dihydroderivative by dihydropyrimidine dehydrogenase. In the second step, dihydropyrimidinase hydrolyzes cleavage of the dihydropyrimidine rings to -ureido compounds. In the third step, /1-ureidopropionase hydrolyzes the j3-ureido compounds to -alanine or fi-aminoisobutyrate (BAIB), with release of ammonia and carbon dioxide. Thus, the major end product of the catabolism of cytosine and uracil is /i-alanine, whereas that of thymine is BAIB. 

D-Amino acids, used as intermediates for semisynthetic penicillins and cephalosporins, have been obtained by stereospecific enzyme-catalyzed hydrolysis of DL-hydantoins.312 The enzymes have been described as hydantoinase and W-carbamoyl-D-amino acid amidohydrolase,313 although some authors identify the former with dihydropyrimidinase.314... 

The six-membered ring systems 5,6-dihydropyrimidine, 5,6-dihydrouracil and 5,6-dihydrothymine can be hydrolyzed by the enzyme dihydropyrimidinase (E.C. 3.5.2.2), which is involved in the degradation of pyrimidine nucleotides. This widely spread, inducible catabolic enzyme is strictly D-selective in contrast to the L-selective dihydroorotase (E. C. 3.5.2.3), which is involved in the opposite anabolic pathway (see above). Another name often used in the literature for the dihydropyrimidinase is d-hydantoinase, because it is also able to hydrolyze D,L-5-monosubstituted hydantoin derivatives with high activity. Both reactions are shown in Fig. 12.4-7. 

Natural cyclic amides such as 5,6-dihydrouracil, uracil and 5,6-dihydrothymine as well as hydantoin, 5-methylhydantoin and 5-hydroxymethylhydantoin are effective inducers for enzyme biosynthesis (for a more detailed review on induction experiments see reference13 ). In some cases, the dihydropyrimidinase (D-hydantoinase) is associated with an N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) and a hydantoin racemase1301. The previously proposed identity of the D-N-carbamoylase with the p-ureidopropionase (E. C. 3.5.1.6), which was assumed to be responsible for the hydrolysis of N-carbamoyl-P-alanine (see Fig. 12.4-7) 131-351 is no longer valid since the investigations of Ogawa et al. on different aerobic bacteria showed that the... 

Figure 12.4-7. Analogy between dihydropyrimidinase- and D-hydantoinase-catalyzed reactions.

L-specific carbamoylase from Pseudomonas putida IFO 12996 also hydrolyzes P-ureidopropionate 14, 36. The enzyme from Pseudomonas putida IFO 12996 was shown to be strictly L-selective and to be active on L-N-formyl- and also on L-N-acetyl-alanine 36. In this context it may be of interest that Runser and Meyer described a d-hydantoinase with no dihydropyrimidinase activity 37 and Ogawa et al. reported on the occurrence of a D-N-carbamoylase with no relation to a D-hydantoinase 38. ... 

Nevertheless, the dihydropyrimidinase seems to be closely related to the barbitur-ase (E.C. 3.5.2.1), which is able to hydrolyze barbituric acid 391 (Fig. 12.4-8). 

Schnackerz were able to show that beef liver dihydropyrimidinase is also able to hydrolyze barbituric acid, although only with low activity140. ... 

Since the early 1950s it has been known that the inducible catabolic enzyme dihydropyrimidinase (E.C. 3.5.2.2) plays an important role in pyrimidine metabolism123, 31, 33, 39, 64-661 and is widespread in nature. The natural substrates of this enzyme, which were also reported to be inducers, are 5,6-dihydrouracil and 5,6-dihy-drothymine. Both compounds are important intermediates in the degradation of pyrimidine nucleotides. The dihydropyrimidinase-reaction is described to be strictly D-specific and to have a wide substrate specificity (see Fig. 12.4-11). In 1970 and... 

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