Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate.
We have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.
Tremblay
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Centre d'Etude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Boul. de l'Université, Sherbrooke (Quebec) J1K 2R1 Canada. rbrzezin@courrier.usherb.ca
Yamaguchi
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Fukamizo
T
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Brzezinski
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R
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D002686:Chitin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D002688:Chitinases / Q000378:metabolism; D048271:Chitosan; D017124:Conserved Sequence; D005944:Glucosamine / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology; D006358:Hot Temperature; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D017510:Protein Folding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism; D013816:Thermodynamics; D014780:Viruses / Q000201:enzymology
D009844:Oligosaccharides; D002686:Chitin; D001224:Aspartic Acid; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase; D002688:Chitinases; D005944:Glucosamine; D000117:Acetylglucosamine