We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates' diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD(+) likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes.
The Entner-Doudoroff (ED) pathway is a classic central pathway of D-glucose metabolism in all three phylogenetic domains. On the other hand, Archaea and/or bacteria possess several modified versions of the ED pathway, in which nonphosphorylated intermediates are involved. Several fungi, including Pichia stipitis and Debaryomyces hansenii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. Gene cluster related to this hypothetical pathway was identified by bioinformatic analysis using the metabolic enzymes involved in analogous sugar pathways to the ED pathway. Furthermore, the homologous gene cluster was found not only in many other fungi but also several bacteria, including Azotobacter vinelandii. Four putative metabolic genes, LRA1-4, were cloned, overexpressed in Escherichia coli, and purified. Substrate specificity and kinetic analysis revealed that nonphosphorylated intermediates related to L-rhamnose are significant active substrates for the purified LRA1-4 proteins. Furthermore, L-2-keto-3-deoxyrhamnonate was structurally identified as both reaction products of dehydration by LRA3 and aldol condensation by LRA4. These results suggested that the LRA1-4 genes encode L-rhamnose 1-dehydrogenase, L-rhamnono-␥-lactonase, L-rhamnonate dehydratase, and L-KDR aldolase, respectively, by which L-rhamnose is converted into pyruvate and L-lactaldehyde through analogous reaction steps to the ED pathway. There was no evolutionary relationship between L-KDR aldolases from fungi and bacteria.The Embden-Meyerhof-Parnas pathway is a central metabolic pathway and is present, at least in part, in all organisms. Furthermore, Gram-negative bacteria possess the Entner-Dou- 2). Schematic sugar conversion is almost analogous to that of the ED pathway, whereas their equivalent metabolic enzymes possess no evolutionary relationship (3-5). Recently, several alternative and/or novel pathways related to D-arabinose (6), L-arabinose (7-10), D-xylose (11, 12), and L-fucose (13), differing from these well known pathways, were identified genetically and were partially analogous to ED and npED pathways. These nonphosphorylative sugar metabolic pathways are classified into two groups, in which sugar is commonly converted into 2-keto-3-deoxyacidsugar through the participation of dehydrogenase, lactonase, and dehydratase. In the "type I pathway" of D-galactose (14), D-fucose (15-18), and L-arabinose (19), 2-keto-3-deoxyacidsugar is cleaved through an aldolase reaction to aldehyde and pyruvate as well as the ED and archaeal npED pathways, although most metabolic genes have not yet been identified. In the bacterial D-gluconate pathway (20), D-gluconate enters the pentose-phosphate pathway (PPP in Fig. 1). In the recently identified bacterial L-fucose pathway (13), an intermediate of L-2-keto-3-deoxyfuconate is dehydrogenated and then cleaved to L-lactate and pyruvate. On the other hand, the "type II pathway" of the nonphosphorylative sugar metabolic pathway corresponds to an alternative...
Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrPC) of PrP into an abnormal form (PrPSc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrPC is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrPSc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrPC, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.
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