title: A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding.
abstract: BACKGROUND
  Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42.
  
  
  RESULTS
  Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity.
  
  
  CONCLUSIONS
  Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan.
authors.affiliation: Département de Biologie, Centre d'Étude et de Valorisation de la Diversité Microbienne, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada. Ryszard.Brzezinski@USherbrooke.ca.
authors.forename: Marie-Ève
authors.initials: MÈ
authors.lastname: Lacombe-Harvey
authors.forename: Mélanie
authors.initials: M
authors.lastname: Fortin
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
authors.forename: Thomas
authors.initials: T
authors.lastname: Letzel
authors.forename: Ryszard
authors.initials: R
authors.lastname: Brzezinski
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D048271:Chitosan / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D048271:Chitosan; D001120:Arginine; D006026:Glycoside Hydrolases; C019868:chitosanase
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