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Structure‐guided analysis of catalytic specificity of the abundantly secreted chitosanase SACTE_5457 from Streptomyces sp. SirexAA‐E
Author(s) -
Takasuka Taichi E.,
Bianchetti Christopher M.,
Tobimatsu Yuki,
Bergeman Lai F.,
Ralph John,
Fox Brian G.
Publication year - 2014
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24491
Subject(s) - chitosanase , glycoside hydrolase , chemistry , stereochemistry , hydrolase , hydrolysis , glucosamine , glycosyl , streptomyces , active site , steric effects , docking (animal) , enzyme , biochemistry , bacteria , biology , genetics , medicine , nursing
SACTE_5457 is secreted by Streptomyces sp. SirexAA‐E, a highly cellulolytic actinobacterium isolated from a symbiotic community composed of insects, fungi, and bacteria. Here we report the 1.84 Å resolution crystal structure and functional characterization of SACTE_5457. This enzyme is a member of the glycosyl hydrolase family 46 and is composed of two α‐helical domains that are connected by an α‐helical linker. The catalytic residues (Glu74 and Asp92) are separated by 10.3 Å, matching the distance predicted for an inverting hydrolysis reaction. Normal mode analysis suggests that the connecting α‐helix is flexible and allows the domain motion needed to place active site residues into an appropriate configuration for catalysis. SACTE_5457 does not react with chitin, but hydrolyzes chitosan substrates with an ∼4‐fold improvement in k cat / K M as the percentage of acetylation and the molecular weights decrease. Analysis of the time dependence of product formation shows that oligosaccharides with degree of polymerization <4 are not hydrolyzed. By combining the results of substrate docking to the X‐ray structure and end‐product analysis, we deduce that SACTE_5457 preferentially binds substrates spanning the −2 to +2 sugar binding subsites, and that steric hindrance prevents binding of N ‐acetyl‐ d ‐glucosamine in the +2 subsite and may weakly interfere with binding of N ‐acetyl‐ d ‐glucosamine in the +1 subsites. A proposal for how these constraints account for the observed product distributions is provided. Proteins 2014; 82:1245–1257. © 2013 Wiley Periodicals, Inc.