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Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115

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Abstract

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

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Abbreviations

BacillusUGL:

Bacillus sp. strain GL1 UGL

ClostridiumUGL:

Clostridium perfringens UGL

C∆6S:

Unsaturated chondroitin disaccharide sulfated at the C-6 position of N-acetylgalactosamine residue

GAG:

Glycosaminoglycan

GH:

Glycoside hydrolase family

PDB:

Protein Data Bank

SagUGL:

Streptococcus agalactiae UGL

UGL:

Unsaturated glucuronyl hydrolase

WT:

Wild type

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Acknowledgments

We thank Drs. S. Baba and N. Mizuno of the Japan Synchrotron Radiation Research Institute (JASRI) for kind help in data collection. Diffraction data for crystals were collected at the BL-38B1 station of SPring-8 (Hyogo, Japan) with the approval of JASRI (Projects 2009A1179, 2010B1149, 2011A1186, 2011B2055, and 2013B1260). We also thank Mss. C. Tokunaga and A. Matsunami for excellent technical assistance. This work was supported in part by Grants-in-Aid from Japan Society for the Promotion of Science (to K. M. and W. H.), a Research Grant (to W.H.) from Mizutani Foundation for Glycoscience, and a research fellowship from Japan Society for the Promotion of Science for Young Scientists (to Y. N.).

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Author notes

  1. Yusuke Nakamichi

    Present address: Laboratory of Supramolecular Crystallography, Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan

Authors and Affiliations

  1. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan

    Yusuke Nakamichi, Sayoko Oiki, Kousaku Murata & Wataru Hashimoto

  2. Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan

    Bunzo Mikami

  3. Department of Life Science, Faculty of Science and Engineering, Setsunan University, Neyagawa, Osaka, 572-8508, Japan

    Kousaku Murata

Authors
  1. Yusuke Nakamichi
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  2. Sayoko Oiki
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Correspondence to Wataru Hashimoto.

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Nakamichi, Y., Oiki, S., Mikami, B. et al. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115. Protein J 35, 300–309 (2016). https://doi.org/10.1007/s10930-016-9673-y

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  • DOI: https://doi.org/10.1007/s10930-016-9673-y

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