Dihydroorotate dehydrogenase reaction

Lieberman and Komberg 11) established the dihydroorotate dehydrogenase reaction in Z. oroticwm as... 

Blattmann P, Retey J (1972) Stereospecificity of the dihydroorotate-dehydrogenase reaction. Eur J Biochem 30 130-137... 

The second step in pyrimidine synthesis is the formation of car-bamoylaspartate, catalyzed by aspartate transcarbamoylase. The pyrimidine ring is then closed hydrolytically by dihydroorotase. Thi resulting dihydroorotate is oxidized to produce orotic acid (onotate, Figure 22.21). The enzyme that produces orotate, dihydroorotate dehydrogenase, is located inside the mitochondria. All other reactions in pyrimidine biosynthesis are cytosolic. [Note The first three enzymes in this pathway (CPS II, aspartate transcarbamoylase, and dihydroorotase) are all domains of the same polypeptide chain. (See k p. 19 for a discussion of domains.) This is an example of a multifunctional or multicatalytic polypeptide that facilitates the ordered synthesis of an important compound.]... 

The third reaction is the oxidation of the ring to form a carbon-carbon bond. The reducing equivalents are transferred to a flavin cofactor of the enzyme dihydroorotate dehydrogenase. The product is orotic acid. 

Dihydroorotate dehydrogenase, the enzyme catalyzing the dehydrogenation of dihydroorotate to orotate (reaction 4 of the pathway Fig. 15-15), is located on the outer side of the inner mitochondrial membrane. This enzyme has FAD as a prosthetic group and in mammals electrons are passed to ubiquinone. The de novo pyrimidine pathway is thus compartmentalized dihydroorotate synthesized by trifunctional DHO synthetase in the cytosol must pass across the outer mitochondrial membrane to be oxidized to orotate, which in turn passes back to the cytosol to be a substrate for bifunctional UMP synthase. Mammalian cells contain two carbamoyl phosphate synthetases the glutamine-dependent enzyme (CPSase II) which is part of CAD, and an ammonia-dependent enzyme (CPSase /) which is found in the mitochondrial matrix, and which is used for urea and arginine biosynthesis. Under certain conditions (e.g., hyperammonemia), carbamoyl phosphate synthesized in the matrix by CPSase I may enter pyrimidine biosynthesis in the cytosol. 

Figure 27-27), aspartate transcarbamoylase, and dihydroorotase activity. Each subunit of Pyr 1-3 has a molecular weight of 200,000-220,000, and the native enzyme exists as multiples of three subunits. The second gene codes for dihydroorotate dehydrogenase which is located on the outer side of the inner mitochondrial membrane. Dihydroorotate, the product of Pyr 1-3, passes freely through the outer mitochondrial membrane and converted to orotate. Orotate readily diffuses to the cytosol for conversion to UMP. The third gene codes for another multifunctional polypeptide known as UMP synthase (Pyr 5,6). Pyr 5,6 (M.W. 55,000) contains orotate phosphoribosyltransferase and orotidylate (orotidine-5 -monophosphate) decarboxylase activity. Use of multifunctional polypeptides is very efficient, since the intermediates neither accumulate nor become consumed in side reactions. They are... 

Conversion of dihydroorotate to orotate is catalyzed by dihydroorotate dehydrogenase, a metalloflavoprotein that contains nonheme-iron atoms and flavin adenine nucleotides (FMN and FAD). In this reaction, the electrons are probably transported via iron atoms and flavin nucleotides that are reoxidized by NAD+. 

Dihydroorotate dehydrogenase (DHOD), dihydropyrimidine dehydrogenase (DPD), and dihydrouridine synthase (DUS), flavoenzymes involved in various stages of pyrimidine metabolism, catalyze very similar C-C oxidation/reduction reactions. These enzymes share common active site residues and structural homology. They have all been implicated as potential drug targets due to their important physiological roles. 

In the next step of pyrimidine biosynthesis, the entire aspartate molecule adds to carbamoyl phosphate in a reaction catalyzed by aspartate transcarbamoylase. The molecule subsequently closes to produce a ring (catalyzed by dihydroorotase), which is oxidized to form orotic acid (or its anion, orotate) through the actions of dihydroorotate dehydrogenase. The enzyme orotate phosphoribosyl transferase catalyzes the transfer of ribose 5-phosphate from PRPP to orotate, producing orotidine 5 -phosphate, which is decarboxylated by orotidylic acid dehydrogenase to form... 

The reaction of carbamoyl phosphate with aspartate to produce W-carbamo-ylaspartate is the committed step in pyrimidine biosynthesis. The compounds involved in reactions up to this point in the pathway can play other roles in metabolism after this point, A -carbamoylaspartate can be used only to produce pyrimidines—thus the term committed step. This reaction is catalyzed by aspartate transcarbamoylase, which we discussed in detail in Ghapter 7 as a prime example of an allosteric enzyme subject to feedback regulation. The next step, the conversion of A-carbamoylaspartate to dihydroorotate, takes place in a reaction that involves an intramolecular dehydration (loss of water) as well as cyclization. This reaction is catalyzed by dihydroorotase. Dihydroorotate is converted to orotate by dihydroorotate dehydrogenase, with the concomitant conversion of NAD to NADH. A pyrimidine nucleotide is now formed by the reaction of orotate with PRPP to give orotidine-5 -monophosphate (OMP), which is a reaction similar to the one that takes place in purine salvage (Section 23.8). Orotate phosphoribosyltransferase catalyzes this reaction. Finally, orotidine-5 -phosphate decarboxylase catalyzes the conversion of OMP to UMP... 

Enzymes catalysing reactions I and III have been crystallised from bacterial sources. The dihydroorotic dehydrogenase is a flavo- protein which is reoxidised by NAD+. Reaction IV involving inversion of configuration at the C(d atom of the ribose sugar, has been studied extensively by Komberg and associates . 

Dihydroorotase dehydrogenase, the enzyme that catalyzes the dehydrogenation of dihydroorotate to orotate (reaction 4 of the pathway Fig. 14-17), is located on the outer side of the inner mitochondrial membrane. This enzyme has FAD as a prosthetic group, and in mammals electrons are passed from it to ubiquinone (Fig. 10-17). 

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