期刊
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
卷 82, 期 7, 页码 1245-1257出版社
WILEY
DOI: 10.1002/prot.24491
关键词
Streptomyces; chitosanase; chitosan; biomass degradation; X-ray structure; GH46
资金
- DOE Great Lakes Bioenergy Research Center (DOE Office of Science) [BER DE-FC02-07ER64494]
- US Department of Energy, Basic Energy Sciences, Office of Science [W 31 109 ENG-38]
- College of Agricultural and Life Sciences, Department of Biochemistry, the Graduate School of the University of Wisconsin
- Michigan Economic Development Corporation
- Michigan Technology Tri-Corridor [085P1000817]
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 angstrom 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 -helical domains that are connected by an -helical linker. The catalytic residues (Glu74 and Asp92) are separated by 10.3 angstrom, matching the distance predicted for an inverting hydrolysis reaction. Normal mode analysis suggests that the connecting -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 approximate to 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. (c) 2013 Wiley Periodicals, Inc.
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