Structure-guided analysis of catalytic specificity of the abundantly secreted chitosanase SACTE_5457 from Streptomyces sp. SirexAA-E

Authors

  • Taichi E. Takasuka,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin
    2. Great Lakes Bioenergy Research Center, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin
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    • Tachi E. Takasuka and Christopher M. Bianchetti contributed equally to this work.

  • Christopher M. Bianchetti,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin
    2. Great Lakes Bioenergy Research Center, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin
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  • Yuki Tobimatsu,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin
    2. Great Lakes Bioenergy Research Center, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin
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  • Lai F. Bergeman,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin
    2. Great Lakes Bioenergy Research Center, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin
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  • John Ralph,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin
    2. Great Lakes Bioenergy Research Center, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin
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  • Brian G. Fox

    Corresponding author
    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin
    2. Great Lakes Bioenergy Research Center, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin
    • Correspondence to: Brian G. Fox, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706. E-mail: bgfox@biochem.wisc.edu

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ABSTRACT

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 kcat/KM 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.

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