Dihydrouridine synthases (DUSs) are flavin-dependent enzymes that catalyze site-specific reduction of uracils in tRNAs. The mechanism of DUS 2 from Saccharomyces cerevisiae was studied. Previously published turnover rates for this DUS were very low. Our studies show that the catalytic cycle consists of reductive and oxidative half-reactions. The enzyme is reduced by NADPH rapidly but has a very slow oxidative half-reaction using in vitro transcribed tRNA substrates. Using tRNA Leu purified from a DUS 2 knockout strain of yeast we obtained reaction rate enhancements of 600-fold over in vitro transcribed substrates, indicating that other RNA modifications are required for rapid uracil reduction. This demonstrates a previously unknown ordering of modifications and indicates that dihydrouridine formation is a later step in tRNA maturation. We also show that an active site cysteine is important for catalysis, likely in the protonation of uracil during tRNA reduction. Dihydrouridine of modified tRNA from Escherichia coli was also oxidized to uridine showing the reaction to be reversible.Dihydrouridine is one of the most common modified nucleosides present in the tRNAs of eubacteria, eukaryotes, and some archea (1). This modified nucleoside is formed by the reduction of the carbon-carbon double bond in uridine at specific sites in tRNAs, most commonly in the D-loop. Our understanding of the physiological role of dihydrouridine is still in its infancy, but one possibility is a structural role. NMR studies have shown that dihydrouridine increases the flexibility of the RNA backbone to allow alternate conformations because its non-planar structure disrupts stacking (2). In addition, a role for dihydrouridine in conformational flexibility is also suggested by its distribution among different species. The dihydrouridine content of species adapted to cold environments (psychrophiles) is the highest found in nature, whereas thermophilic species tend to have much less or no dihydrouridine (3). It has been suggested that the chemical stability of RNA structures is enhanced by the absence of dihydrouridine, perhaps explaining why thermophilic species often lack dihydrouridine, to avoid hydrolytic ring opening at high temperatures (4).Recent studies have hinted at further physiological roles for dihydrouridine. tRNA lacking dihydrouridine, along with other modifications, degrades at a substantially increased rate, approaching that seen for mRNA (5). The main function of dihydrouridine may therefore be to protect tRNAs from degradation. It has long been known that dihydrouridine levels are increased in cancerous tissues (6). Recent studies have shown that the human dihydrouridine synthase 2 is responsible for the increased dihydrouridine levels formed in pulmonary carcinogenesis (7). This information suggests that dihydrouridine plays an important role in preventing tRNA turnover.The family of enzymes catalyzing the reduction of uridine in tRNA, dihydrouridine synthases (DUS), 3 has recently been identified in Saccharomyces cerevi...

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...

The flavoenzyme dihydroorotate dehydrogenase A from Lactococcus lactis is a homodimeric protein of 311 residues/subunit, and the two active sites are positioned at a distance from the dimer interface. To promote formation of the monomeric form of the enzyme, we changed the residues involved in formation of two salt bridges formed between the residues Glu206 of the one polypeptide and Lys296 of the other polypeptide. The mutant enzymes formed inactive precipitates when cells were grown at 37°C, but remained soluble and active when cells were grown at 25°C. The salt bridges were not needed for activity, because the mutant enzymes in which one of the residues was converted to an alanine (E206A or K296A) retained almost full activity. The mutant enzymes in which the charge of one of the residues of the salt bridge was inverted (i.e., E206K or K296E) were severely impaired. The double mutant E206K-K296E, which has the possibility of forming salt bridges in the opposite orientation of the wild type, was fully active in concentrated solutions, but dissociated into inactive monomers upon dilution. The K D for the dimer to monomer dissociation reaction was 12 M, and dimer formation was favored by the product, orotate, or by high ionic strength, indicating that the hydrophobic interactions are important for the subunit contacts. Wild-type dihydroorotate dehydrogenase A was similarly found to dissociate into inactive monomers, but with a K D for dissociation equal to 0.12 M. These results imply that the dimeric state is necessary for activity of the enzyme.

Background Biosimilars must meet stringent regulatory requirements, both at the time of authorization and during their lifecycle. Yet it has been suggested that divergence in quality attributes over time may lead to clinically meaningful differences between two versions of a biologic. Therefore, this study investigated the batch-to-batch consistency across a range of parameters for released batches of the etanercept biosimilar (SB4) and infliximab biosimilar (SB2). Methods SB4 (Benepali ® ) and SB2 (Flixabi ® ) were both developed by Samsung Bioepis and are manufactured in Europe by Biogen at their facility in Hillerød, Denmark. A total of 120 batches of SB4 and 25 batches of SB2 were assessed for consistency and compliance with specified release parameters, including purity, post-translational glycosylation (SB4 only), protein concentration, and biological activity. Results The protein concentration, purity, tumor necrosis factor-α (TNF-α) binding, and TNF-α neutralization of all batches of SB4 and SB2 were within the strict specification limits set by regulatory agencies, as was the total sialic acid (TSA) content of all batches of SB4. Conclusions Quality attributes of SB4 and SB2 batches showed little variation and were consistently within the rigorous specifications defined by regulatory agencies.As of August 2019, the Biogen Hillerød facility (Denmark) became Fujifilm Diosynth Biotechnologies Denmark ApS, a contract development and manufacturing organization (CDMO) for the manufacture of biosimilars developed by Samsung Bioepis. Key PointsBiosimilars are held to the same rigorous quality standards as any other biologic. SB4 and SB2 biosimilars demonstrated a high degree of batch-to-batch consistency.Quality attributes including purity, percentage of high molecular weight species, tumor necrosis factor-α (TNFα) binding, and TNF-α neutralization remained well within acceptance limits. 2

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