The thiO gene of Bacillus subtilis encodes an FAD-dependent glycine oxidase. This enzyme is a homotetramer with a monomer molecular mass of 42 kDa. In this paper, we demonstrate that ThiO is required ...for the biosynthesis of the thiazole moiety of thiamin pyrophosphate and describe the structure of the enzyme with N-acetylglycine bound at the active site. The closest structural relatives of ThiO are sarcosine oxidase and d-amino acid oxidase. The ThiO structure, as well as the observation that N-cyclopropylglycine is a good substrate, supports a hydride transfer mechanism for the enzyme. A mechanistic proposal for the role of ThiO in thiazole biosynthesis is also described.
The recent H7N9 influenza outbreak in China highlights the need for influenza vaccine production systems that are robust and can quickly generate substantial quantities of vaccines that target new ...strains for pandemic and seasonal immunization. Although the influenza vaccine system, a public-private partnership, has been effective in providing vaccines, there are areas for improvement. Technological advances such as mammalian cell culture production and synthetic vaccine seeds provide a means to increase the speed and accuracy of targeting new influenza strains with mass-produced vaccines by dispensing with the need for egg isolation, adaptation, and reassortment of vaccine viruses. New influenza potency assays that no longer require the time-consuming step of generating sheep antisera could further speed vaccine release. Adjuvants that increase the breadth of the elicited immune response and allow dose sparing provide an additional means to increase the number of available vaccine doses. Together these technologies can improve the influenza vaccination system in the near term. In the longer term, disruptive technologies, such as RNA-based flu vaccines and 'universal' flu vaccines, offer a promise of a dramatically improved influenza vaccine system.
H1N1: can a pandemic cycle be broken? Settembre, Ethan C; Dormitzer, Philip R; Rappuoli, Rino
Science translational medicine,
03/2010, Letnik:
2, Številka:
24
Journal Article
Recenzirano
The influenza virus that caused the 2009 H1N1 swine-origin flu pandemic is antigenically similar to the one that caused the devastating 1918 pandemic. Over time, the human population became ...susceptible to a modified version of the 1918 pandemic H1N1 virus that had been archived in swine. Now, two papers, one in this issue of Science Translational Medicine and one in Science, shed mechanistic light on how glycosylation gave rise to seasonal human flu viruses that are immunologically distinct from their 1918 pandemic precursor and the 2009 pandemic strain. These findings suggest strategies to anticipate and prevent future pandemics.
Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine ...dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four‐stranded parallel β‐sheet flanked by four α‐helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a 4Fe–4S cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii Sakuraba et al. (2005), J. Biol. Chem.280, 26645–26648. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the 4Fe–4S cluster, prior to cluster assembly.
Thiazole synthase is the key enzyme involved in the formation of the thiazole moiety of thiamin pyrophosphate. We have determined the structure of this enzyme in complex with ThiS, the sulfur carrier ...protein, at 3.15 Å resolution. Thiazole synthase is a tetramer with 222 symmetry. The monomer is a (βα)8 barrel with similarities to the aldolase class 1 and flavin mononucleotide dependent oxidoreductase and phosphate binding superfamilies. The sulfur carrier protein (ThiS) is a compact protein with a fold similar to that of ubiquitin. The structure allowed us to model the substrate, deoxy-d-xylulose 5-phosphate (DXP), in the active site. This model identified Glu98 and Asp182 as new active site residues likely to be involved in the catalysis of thiazole formation. The function of these residues was probed by mutagenesis experiments, which confirmed that both residues are essential for thiazole formation and identified Asp182 as the base involved in the deprotonation at C3 of the thiazole synthase DXP imine. Comparison of the ThiS binding surface to the surface of ubiquitin identified a conserved hydrophobic patch of unknown function on ubiquitin that may be involved in complex formation between ubiquitin and one of its binding partners.
Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2′-deoxyuridine to ...2′-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2′-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2′. Crystals of bovine uridine phosphorylase treated with 2′-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.
The development of new and effective antiprotozoal drugs has been a difficult challenge because of the close similarity of the metabolic pathways between microbial and mammalian systems. ...5‘-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is thought to be an ideal target for therapeutic drug design as the enzyme is present in many microbes but not in mammals. MTA/AdoHcy nucleosidase (MTAN) irreversibly depurinates MTA or AdoHcy to form adenine and the corresponding thioribose. The inhibition of MTAN leads to a buildup of toxic byproducts that affect various microbial pathways such as quorum sensing, biological methylation, polyamine biosynthesis, and methionine recycling. The design of nucleosidase-specific inhibitors is complicated by its structural similarity to the human MTA phosphorylase (MTAP). The crystal structures of human MTAP complexed with formycin A and 5‘-methylthiotubercidin have been solved to 2.0 and 2.1 Å resolution, respectively. Comparisons of the MTAP and MTAN inhibitor complexes reveal size and electrostatic potential differences in the purine, ribose, and 5‘-alkylthio binding sites, which account for the substrate specificity and reactions catalyzed. In addition, the differences between the two enzymes have allowed the identification of exploitable regions that can be targeted for the development of high-affinity nucleosidase-specific inhibitors. Sequence alignments of Escherichia coli MTAN, human MTAP, and plant MTA nucleosidases also reveal potential structural changes to the 5‘-alkylthio binding site that account for the substrate preference of plant MTA nucleosidases.
Calls to develop a universal influenza vaccine have increased in the wake of the 2009 H1N1 influenza pandemic. This demand comes at a time when analyses of the human antibody repertoire, informed by ...structures of complexes between broadly neutralizing antibodies and influenza hemagglutinin, have revealed the target of a class of broadly neutralizing antibodies. Recent studies suggest a path forward to more broadly protective influenza vaccines.
Uridine phosphorylase (UP) catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1‐phosphate and is a key enzyme in the pyrimidine‐salvage pathway. Escherichia coli UP is ...structurally homologous to E. coli purine nucleoside phosphorylase and other members of the type I family of nucleoside phosphorylases. The structures of 5‐benzylacyclouridine, 5‐phenylthioacyclouridine, 5‐phenylselenenylacyclouridine, 5‐m‐benzyloxybenzyl acyclouridine and 5‐m‐benzyloxybenzyl barbituric acid acyclonucleoside bound to the active site of E. coli UP have been determined, with resolutions ranging from 1.95 to 2.3 Å. For all five complexes the acyclo sugar moiety binds to the active site in a conformation that mimics the ribose ring of the natural substrates. Surprisingly, the terminal hydroxyl group occupies the position of the nonessential 5′‐hydroxyl substituent of the substrate rather than the 3′‐hydroxyl group, which is normally required for catalytic activity. Until recently, inhibitors of UP were designed with limited structural knowledge of the active‐site residues. These structures explain the basis of inhibition for this series of acyclouridine analogs and suggest possible additional avenues for future drug‐design efforts. Furthermore, the studies can be extended to design inhibitors of human UP, for which no X‐ray structure is available.
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