Conformational Change in the Active Site of Strept

Authors: Yusuke Nakamichi Sayoko Oiki Bunzo Mikami Kousaku Murata Wataru Hashimoto

Publish Date: 2016/07/11

Volume: 35, Issue: 4, Pages: 300-309

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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 Asp115 and Asp175 of Streptococcus agalactiae UGL SagUGL are completely conserved in other bacterial UGLs one of which Asp175 of SagUGL acts as a general acid and base catalyst The other Asp Asp115 of SagUGL also affects the enzyme activity although its role in the enzyme reaction has not been well understood Here we show substitution of Asp115 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 200 and 179 Å resolution respectively by Xray crystallography The side chain of Asn115 is drastically shifted in both mutants owing to the interaction with several residues including Asp175 by formation of hydrogen bonds This interaction between Asn115 and Asp175 probably prevents the mutants from triggering the enzyme reaction using Asp175 as an acid catalystWe 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 BL38B1 station of SPring8 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 GrantsinAid from Japan Society for the Promotion of Science to K M and W H a Research Grant to WH 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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