Journal
FEBS JOURNAL
Volume 282, Issue 20, Pages 4001-4014Publisher
WILEY
DOI: 10.1111/febs.13401
Keywords
Actinomycete; chimeric enzyme; galactosylmannooligosaccharide; glycoside hydrolase family 5; mannanase
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Funding
- Grants-in-Aid for Scientific Research [26850129, 14J04026, 26450114] Funding Source: KAKEN
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Endo--1,4-mannanases from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) demonstrated different substrate specificities. StMan hydrolyzed galactosylmannooligosaccharide (GGM5; 6(III),6(IV)--d-galactosyl mannopentaose) to GGM3 and M2, whereas TfMan hydrolyzed GGM5 to GGM4 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 angstrom, 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 GH5 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.
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