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The loop structure of Actinomycete glycoside hydrolase family 5 mannanases governs substrate recognition
Author(s) -
Kumagai Yuya,
Yamashita Keitaro,
Tagami Takayoshi,
Uraji Misugi,
Wan Kun,
Okuyama Masayuki,
Yao Min,
Kimura Atsuo,
Hatanaka Tadashi
Publication year - 2015
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13401
Subject(s) - hydrolase , glycoside hydrolase , substrate (aquarium) , stereochemistry , hydrolysis , chemistry , mutant , galactose , enzyme , biochemistry , crystallography , biology , ecology , gene
Endo‐β‐1,4‐mannanases from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) demonstrated different substrate specificities. StMan hydrolyzed galactosylmannooligosaccharide ( GGM 5; 6 III ,6 IV ‐α‐ d ‐galactosyl mannopentaose) to GGM 3 and M2, whereas TfMan hydrolyzed GGM 5 to GGM 4 and M1. To determine the region involved in the substrate specificity, we constructed chimeric enzymes of StMan and TfMan and evaluated their substrate specificities. Moreover, the crystal structure of the catalytic domain of StMan (StMandC) and the complex structure of the inactive mutant StE273AdC with M6 were solved at resolutions of 1.60 and 1.50 Å, respectively. Structural comparisons of StMandC and the catalytic domain of TfMan lead to the identification of a subsite around −1 in StMandC that could accommodate a galactose branch. These findings demonstrate that the two loops (loop7 and loop8) are responsible for substrate recognition in GH 5 actinomycete mannanases. In particular, Trp281 in loop7 of StMan, which is located in a narrow and deep cleft, plays an important role in its affinity toward linear substrates. Asp310 in loop8 of StMan specifically bound to the galactosyl unit in the −1 subsite.