232 I. LIEBERMAN, A, KORNBERG VOL. 12 (1953) 



DISCUSSION 



The first step in the metabohsm of orotic acid by this anaerobic bacterium is a 

 reduction involving the utihzation of one mole of DPNH. The crystalline product isolated 

 from this reaction has an elementary analysis and equivalent weight compatible with 

 dihydro-orotic acid. Since this natural product is distinguishable from the compound 

 synthesized by the fusion of maleic acid and urea and also claimed to be dihydro-orotic 

 acid^", it is possible that two isomeric forms are represented. Unlike the synthetic com- 

 pound, the natural product is rapidly and quantitatively oxidized to orotic acid by the 

 bacterial enzyme preparation and serves as a growth factor for Lactobacillus bulgaricus 

 og. The latter observation provides some evidence that the enzymic reduction of orotic 

 acid takes place at the C4-C5 bond since ureidosuccinic acid and orotic acid were shown 

 to be the only effective pyrimidine nucleotide precursors for the bacterium', and a 

 dihydro-orotic acid of this configuration is a logical intermediate between these two 

 compounds. Further evidence for reduction of the C4-C5 bond is the observed enzymic 

 interconversion of orotic acid and ureidosuccinic acid^^ in which this form of dihydro- 

 orotic acid again represents the most plausible intermediate. 



These findings which establish a reduction product of a pyrimidine as the immediate 

 metabolic precursor (and derivative) of the pyrimidine do not appear to be limited 

 to the activities of this anaerobic bacterium. Rather, they are consistent with the findings 

 of other workers that the reduced forms of uracil and thymine are metabolically active. 

 Fink ^^ al.^^ have demonstrated that rat liver slices convert large quantities of dihydro- 

 thymine and dihydrouracil to j8-amino/sobutyric acid and ^-alanine, respectively. While 

 thymine and uracil were relatively inert in their system, it seems likely, as they suggest, 

 that the pathways of thymine and uracil metabolism involve the reduced derivatives 

 which were not produced by the in vitro system used. The isolation of dihydrouracil 

 from beef spleen^^ and its active utilization as a nitrogen source by Torula utilis^'^ rnay 

 be taken as additional evidence for regarding the dihydropyrimidines as important 

 metabolic intermediates. The finding^" that the maleic acid-urea fusion product^" is the 

 only pyrimidine derivative metabolized by Saccharomyccs cerevisiae is difficult to evaluate 

 in view of the uncertainty regarding its structure. 



So little is known about the mechanism of the biological synthesis of pyrimidine 

 nucleotides that it is impossible to determine the validity of the general impression that 

 orotic acid serves as a direct precursor. It is conceivable, for example, that dihydro- 

 orotic rather than orotic acid may be the immediate precursor of nucleic acid pyrimidines. 

 The availability of dihydro-orotic acid may now facilitate attempts to resolve this and 

 related questions. 



SUMMARY 



1. An enzyme was partially purified from extracts of an anaerobic soil bacterium isolated by 

 enrichment culture on orotic acid. The enzyme, named dihydro-orotic dehydrogenase, was shown to 

 catalyze the reaction : 



Orotate + DPNH + H+ ^ Dihydro-orotate -j- DPN. 



2. The product of the enzymic reduction of orotic acid was isolated in crystalline form and shown 

 to have the correct elementary analysis and equivalent weight for dihydro-orotic acid; it is provision- 

 ally considered to be a dihydro-orotic acid saturated at the C4-C5 bond. 



3. The ecjuilibrium of the reaction strongly favours orotic acid reduction; oxidation of dihydro- 

 orotic acid was demonstrable only with the aid of a system to remove DPNH. • 



References p. 234. 



