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Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme

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Abstract

Glycogen debranching enzyme (GDE) is bifunctional in that it exhibits both 4-α-glucanotransferase and amylo-α-1,6-glucosidase activity at two distinct catalytic sites. GDE converts the phosphorylase-limit biantennary branch [G-G-G-G-(G-G-G-G↔)G-G- residue, where G = d-glucose, hyphens represent α-1,4-glycosidic bonds, and the double-headed arrow represents an α-1,6-glycosidic bond] into a linear maltooligosyl residue, which is then subjected to phosphorylase, and glycogen degradation continues. The prevailing hypothesis regarding the glycogen debranching pathway was that 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch into the G-G-G-G-G-G-G-(G↔)G-G- residue and amylo-α-1,6-glucosidase cleaves the remaining α-1,6-linked G residue. In the present study, we analyzed the substrate specificities of 4-α-glucanotransferase and amylo-α-1,6-glucosidase using fluorogenic biantennary dextrins such as G-G-G-G-(G-G-G-G↔)G-G-GPA (F4/4/2; where GPA = 1-deoxy-1-[(2-pyridyl)amino]-d-glucitol), G-(G-G-G-G↔)G-G-GPA (F1/4/2), and G-G-G-G-G-G-G-(G↔)G-G-GPA (F7/1/2). Contrary to the prevailing hypothesis, the main branch of F4/4/2 was an important donor substrate component of 4-α-glucanotransferase and did not serve as an acceptor substrate. However, when G-G-G-G-G-GPA was added to the mixture, it successfully accepted a maltotriosyl (G3-) residue from F4/4/2. In addition, amylo-α-1,6-glucosidase exhibited strong activity towards G-G-G-G-(G↔)G-G-GPA but weak activity towards F7/1/2. Furthermore, the debranching activity of GDE towards phosphorylase-limit glycogen substantially increased when methyl α-maltooligosides with lengths equal to or greater than that of methyl α-maltopentaoside (G5-OCH3) were added to the enzyme reaction mixture. Based on these results, we propose the following macroscopic debranching pathway: Via 4-α-glucanotransferase, the G3- residue of the donor branch is transferred to a long (n ≥ 5) linear Gn- residue linked to a different branching G residue.

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Data supporting the findings of the present study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank Prof. Takaaki Miyaji and Ms. Asako Kawakami at the Department of Genomics & Proteomics of the Advanced Science Research Center of Okayama University for performing MALDI-TOF MS on the fluorogenic biantennary dextrins.

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The authors did not receive support from any organization for the submitted work.

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  1. Ayato Ikeda and Yasushi Makino contributed equally to this work.

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  1. Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Naka-ku, Sakai, Osaka, 599-8531, Japan

    Ayato Ikeda, Yasushi Makino & Hiroshi Matsubara

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  1. Ayato Ikeda
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  2. Yasushi Makino
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  3. Hiroshi Matsubara
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Correspondence to Yasushi Makino.

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Ikeda, A., Makino, Y. & Matsubara, H. Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme. Glycoconj J 39, 345–355 (2022). https://doi.org/10.1007/s10719-022-10046-y

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