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Glycogen storage diseases: introduction
Published in William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop, Atlas of Inherited Metabolic Diseases, 2020
William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop
The phosphorolysis of glycogen catalyzed by phosphorylase splits off glucose units until the 1,6 branch points are approached. These branches are degraded down to a limit dextrin in which three glucose residues are attached in 1,4 linkage to the 1,6-linked glucose. The transfer of this trisaccharide to the end of another glycogen chain is catalyzed by the transferase activity of the debranching enzyme. Then, the exposed glucose at the branch point is cleaved by the same enzyme protein in which amylo-1,6-glucosidase activity is at a different catalytic site [13]. The product of the reaction is free glucose. The combined activity of phosphorylase and the debranching enzyme accomplish the complete degradation of glycogen.
RNA
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Further development of the Qβ sequencing occurred in the obstacles of the gene engineering approaches. As in the case of the MS2 RNA, the Qβ RNA was extended at the 3′-end with the polyA segment by terminal riboadenylate transferase purified from calf thymus (Gilvarg et al. 1974, 1975a) The polyadenylated Qβ RNA retained full infectivity in a spheroplast assay system. However, the progeny viruses did not contain polyA termini, indicating an in vivo rectification of the in vitro alteration (Gilvarg et al. 1975a). While the polyA-Qβ RNA functioned normally as messenger for the synthesis of virus-specific proteins, it had lost its capacity to serve as template for the Qβ replicase. The template function was restored, however, by phosphorolysis with polynucleotide phosphorylase. It was concluded that a host enzyme, perhaps polynucleotide phosphorylase, removed part or all of the adenylate residues prior to replication of the RNA in vivo (Gilvarg et al. 1975b). The Qβ RNA was elongated also with a 3′-terminal oligoC tract (Mekler and Billeter 1975). Meanwhile, polyA sequences were added to the 3′-terminus of the Qβ RNA by ATP:RNA adenylyltransferase from E. coli by Fiers’ team (Devos et al. 1976c). By tail lengths not exceeding 200 nucleotide residues, the physical properties of Qβ-RNA-polyA were only slightly different from those of the original RNA, but almost complete abolishment of template activity, even by short oligoA stretches, was found, in agreement with the conclusions of Weissmann’ team.
Developmental Aspects of the Alveolar Epithelium and the Pulmonary Surfactant System
Published in Jacques R. Bourbon, Pulmonary Surfactant: Biochemical, Functional, Regulatory, and Clinical Concepts, 2019
Jacques R. Bourbon, Caroline Fraslon
In order to further explore the significance of glycogen utilization through the hydrolytic process, Bourbon and co-workers208 have used an inhibitor of acid a-glucosidase, the pseudosaccharide acarbose. The latter simultaneously reduced glycogenolysis and surfactant-phospholipid synthesis in fetal rat lung expiants when added to culture medium. By contrast, acarbose did not change significantly the synthesis of extrasurfactant phospholipids. It therefore appears that the part of glycogen stores which is derived from surfactant phospholipid anabolism must be primarily degraded through the hydrolytic process, which, as stated above, would be under adrenocortical control. Phosphorolysis of glycogen would possibly provide substrates for other utilizations through the glycolytic and hexose monophosphate pathways. The participation of lamellar bodies themselves is suggested by the presence of α-glucosidase activities in these organelles, especially the specific isoenzyme observed by de Vries and co-workers,207 and by the observation in the developing mouse26 and monkey225 lung of glycogen particles inside electron-dense pregranules and the immature lamellar bodies.
Interactions of 2,6-substituted purines with purine nucleoside phosphorylase from Helicobacter pylori in solution and in the crystal, and the effects of these compounds on cell cultures of this bacterium
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Marta Narczyk, Marta Ilona Wojtyś, Ivana Leščić Ašler, Biserka Žinić, Marija Luić, Elżbieta Katarzyna Jagusztyn-Krynicka, Zoran Štefanić, Agnieszka Bzowska
Usually, the specific activity of PNP is determined with inosine as a substrate, using the assay in which the product of phosphorolysis, hypoxanthine, is oxidised to uric acid in a coupled reaction catalysed by xanthine oxidase27. However, for inhibition studies of PNP from H. pylori strain 26695, 7-methylguanosine and guanosine were used as nucleoside substrates to avoid possible inhibition of xanthine oxidase by the tested compounds. Phosphorolysis of these two substrates may be observed using a direct spectrophotometric method as described previously28,29. Spectral data for substrate concentration determination and for the activity assays used in this study, are the following: εmax =13,650 M−1 cm−1 for Guo29, εmax = 8500 M−1 cm−1 for m7Guo (at pH 7.0)28, Δε = −4850 M−1 cm−1 at λobs = 252 nm for Guo29 and Δε = −4600 M−1 cm−1 at λobs = 258 nm for m7Guo (at pH 7.0)28.
Advances in biocatalytic and chemoenzymatic synthesis of nucleoside analogues
Published in Expert Opinion on Drug Discovery, 2022
Sebastian C. Cosgrove, Gavin J. Miller
The chemical synthesis of 4’-modified nucleosides is challenging and lengthy, requiring multiple steps and protecting group manipulations and is often molecule specific, presenting little opportunity for scaffold diversification. In 2021, the Kurreck group demonstrated a biocatalytic approach, using a pyrimidine nucleoside phosphorylase (PyNP) from Thermus thermophilus (TtPyNP), to divergently access 4’-methylated nucleosides (Scheme 2) [14]. This approach compliments a scalable chemical synthesis of nucleosides (which included 4’-modifications) recently reported by Britton and colleagues [15]. Nucleoside phosphorylases catalyze the reversible phosphorolysis of nucleosides to their corresponding pentose 1-phosphate and purine/pyrimidine components. A subsequent reverse phosphorolysis can then incorporate an alternative nucleobase [16]. This transglycosylation effectively interconverts the nucleobase components across a pentose scaffold and was used effectively for 4’-alkynyl installation within the Merck biocatalytic synthesis of Islatravir (vide infra) [17].
Nuclease activity: an exploitable biomarker in bacterial infections
Published in Expert Review of Molecular Diagnostics, 2022
Javier Garcia Gonzalez, Frank J. Hernandez
Furthermore, beyond nuclease homologues, differences in nuclease populations among bacterial species have been reported that suggest that different bacteria may use non-homologous nucleases to fulfill similar biological roles through disparate pathways and mechanisms pointing towards the existence of yet undescribed novel intracellular nucleases across bacterial species. This phenomenon is illustrated by nuclease populations identified in two putative model organisms, namely E. coli and B. subtillis. Despite sharing several conserved homologous nucleases, an even greater number of nucleases are unique for each species. As such, major differences in the mode of action between B. subtillis and E. coli nuclease populations exist. While 90% of the nucleases found in E. coli extracts are hydrolytic, phosphorolysis dominates the nuclease activity in B. subtillis extracts [39]. However, as reported before [26], different catalytic mechanisms are poorly correlated with biological roles, as exemplified by the fact that some of the unique non-homologous nucleases, such as RNase E (E. coli) and RNase Y in (B. subtillis) possess equivalent biological roles [47]. Similarly, several exonucleases belonging to different protein families share their preference for ssDNA and have seemingly redundant functions, as reported for E. coli [38].