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The phosphoribosyltransferases catalyze the formation of nucleotides by reaction with 5-phosphoribosyl-1-diphosphate (PRPP) and a nitrogenous base, releasing pyrophosphate as the second reaction product. The enzymes participate in the biosynthesis of the amino acids histidine and tryptophan, pyridine coenzymes, and all nucleotides by de novo synthesis and salvage pathways [1]. They are generally unregulated enzymes obeying hyperbolic saturation kinetics for the substrates, but there are exceptions. The glutamine PRPP amidotransferase, which catalyses the first step specific for de novo purine nucleotide synthesis, is feedback inhibited by AMP and GMP, and uracil phosphoribosyltransferase (UPRTase; a salvage enzyme that makes UMP from uracil and PRPP) was shown to be activated by GTP in several organisms e.g. The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60°C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number k cat and raised the K m values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two-and >10-fold, respectively. The inhibition by CTP was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously (K D equal to 2 and 0.5 lm, respectively). The binding of each of the inhibitors was incompatible with binding of PRPP or GTP. The data indicate that UPRTase undergoes a transition from a weakly active or inactive T-state, favored by binding of UMP and CTP, to an active R-state, favored by binding of GTP and PRPP.Abbreviations IPTG, isopropyl thio-b-D-galactoside; GdHCl, guanidinium chloride; PP i , inorganic diphosphate or pyrophosphate; PRPP, 5-phosphoribosyl-1-adiphosphate; UPRTase, uracil phosphoribosyltransferase or UMP synthase (EC 2.4.2.9).

Two experiments were performed to study the effect of xylanase on ileal extract viscosity, in vivo fiber solubilization and degradation, and apparent ileal digestibility (AID) of fiber constituents, OM, CP, starch, and crude fat in rye and wheat in ileal-cannulated pigs. In Exp. 1, coarse rye without (NX) or with addition of xylanase from Aspergillus niger (AN), (BS), or (TR) was fed to 8 ileal-cannulated barrows (initial BW 30.9 ± 0.3 kg) for 1 wk each according to a double 4 × 4 Latin square design. In Exp. 2, fine rye, fine wheat, and coarse wheat with or without a combination of xylanase from and were fed to 6 ileal-cannulated barrows (initial BW 33.6 ± 0.5 kg) for 1 wk according to a 6 × 6 Latin square design with a 2 × 3 factorial arrangement of enzyme and cereal matrix. Chromic oxide (0.2%) was used as an inert marker. Ileal effluent was collected for 8 h on d 5 and 7 and pooled for analysis. In Exp. 1, TR reduced intestinal viscosity of pigs fed rye from 9.3 mPa·s in the control diet (NX) to 6.0 mPa·s ( < 0.001), whereas AN and BS had no effect. None of the enzymes changed the concentration of total arabinoxylan, high-molecular-weight arabinoxylan (HMW-AX), or arabinoxylan oligosaccharides (AXOS) in the liquid phase of digesta. In Exp. 2, the enzyme combination reduced intestinal viscosity for all 3 cereal matrices ( < 0.05), but the viscosity was much higher with fine rye (7.6 mPa·s) than with fine and coarse wheat (<1.7 mPa·s). Simultaneously, the total concentration of arabinoxylan in the liquid phase of digesta increased by 82.4% in fine wheat ( < 0.002) and by 45.9% in coarse wheat ( < 0.006), and AXOS increased 16-fold with enzyme addition. Similar effects of enzyme were not seen with rye. The concentration of xylooligosaccharides in the liquid phase of digesta increased with enzyme addition, but for xylose, it was only significant for wheat, for which it increased 3.9-fold ( < 0.001). None of the xylanases affected AID of arabinoxylan of rye in Exp. 1. In Exp. 2, the enzyme combination increased AID of arabinoxylan by 91% to 107% ( < 0.001) across cereal matrices. Enzyme addition did not affect AID of nutrients in any of the experiments except for a higher starch and crude fat digestibility of fine wheat with enzyme addition ( < 0.012) in Exp. 2. Collectively, the results suggest that xylanase is more efficient in degrading arabinoxylan from wheat than from rye.

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Dihydroorotate dehydrogenases (DHODs) are flavoenzymes catalyzing the oxidation of (S)-dihydroorotate to orotate in the biosynthesis of UMP, the precursor of all other pyrimidine nucleotides. On the basis of sequence, DHODs can be divided into two classes, class 1, further divided in subclasses 1A and 1B, and class 2. This division corresponds to differences in cellular location and the nature of the electron acceptor. Herein we report a study of Lactococcus lactis DHODA, a representative of the class 1A enzymes. Based on the DHODA structure we selected seven residues that are highly conserved between both main classes of DHODs as well as three residues representing surface charges close to the active site for site-directed mutagenesis. The availability of both kinetic and structural data on the mutant enzymes allowed us to define the roles individual structural segments play in catalysis. We have also structurally proven the presence of an open active site loop in DHODA and obtained information about the interactions that control movements of loops around the active site. Furthermore, in one mutant structure we observed differences between the two monomers of the dimer, confirming an apparent asymmetry between the two substrate binding sites that was indicated by the kinetic results.Dihydroorotate dehydrogenases (DHODs) 1 catalyze the stereospecific oxidation of (S)-dihydroorotate to orotate through reduction of their prosthetic FMN group. This is the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. In rapidly proliferating cells pyrimidine salvage pathways cannot compensate for the lack of UMP caused by inhibition of DHOD. This makes DHODs attractive targets for antiproliferative, antiparasitic, and immunosuppresive drugs used in organ transplantation and in treatment of inflammatory diseases (1-6).Based on presently known sequences, DHODs can be divided into two main classes (7). Class 1 enzymes, which are subdivided into class 1A and 1B enzymes, are cytosolic proteins, whereas class 2 enzymes are membrane-associated. The bacterium Lactococcus lactis contains genes that encode DHODs representing subclass 1A and 1B, DHODA, and DHODB. Crystal structures have been determined for both of these without and in the presence of the product orotate (8 -10). DHODA is a dimer formed by two identical PyrD subunits each containing an FMN group (11). The natural electron acceptor for DHODA is fumarate (12), but all DHODs can to some extent use a variety of other electron acceptors such as soluble quinones, dyes, and molecular oxygen (13, 14). For DHODA it has been suggested that the substrate and the natural electron acceptor use the same binding site. Kinetic investigations supported a one-site ping-pong mechanism and showed the second halfreaction to be the rate-limiting step for DHODA (13).The class 1B enzyme is a heterotetramer consisting of two PyrDB subunits, homologous to the PyrDA subunits of DHODA, and two PyrK subunits (15). The PyrK subunits each contain FAD and an [2Fe-2S] cluster, and...