title: Structure-guided analysis of catalytic specificity of the abundantly secreted chitosanase SACTE_5457 from Streptomyces sp. SirexAA-E.
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 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.
authors: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, 53706; Great Lakes Bioenergy Research Center, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin, 53726.
authors: Taichi E
authors: TE
authors: Takasuka
authors: Christopher M
authors: CM
authors: Bianchetti
authors: Yuki
authors: Y
authors: Tobimatsu
authors: Lai F
authors: LF
authors: Bergeman
authors: John
authors: J
authors: Ralph
authors: Brian G
authors: BG
authors: Fox
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase
keywords: GH46; Streptomyces; X-ray structure; biomass degradation; chitosan; chitosanase