Abstract
BackgroundStreptomyces 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.ResultsMutation 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.ConclusionsArginine 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.
Highlights
In order to verify if a similar kind of interaction is needed to sustain the catalytic potential of the general base residue, we examined the microenvironment of Asp40 of CsnN174 and found that an arginine (Arg42) is highly conserved among the glycoside hydrolases family 46 (GH46) family of chitosanases
Choice of Arg42 for site-specific saturation mutagenesis To find out if there were any residues that could possibly assist Asp40 to achieve its function as catalytic general base, we first examined the 3D structure of the catalytic cleft of Streptomyces sp
The structure-based alignment of the primary sequence of CsnN174 with the other members of GH46 family revealed that the arginine residue at position 42 is conserved in all the chitosanases biochemically characterized [17] strongly suggesting that this arginine plays an important role in catalysis [3]
Summary
N174 (CsnN174), which belongs to the glycoside hydrolases family 46 (GH46), is among the best characterized [2,3,4] This enzyme is an endo-type hydrolase and proceeds via an inverting mechanism in which Glu acts as the general acid and Asp as the general base/nucleophile [4,5]. The structural features of CsnN174 are shared by GH46 members, and by GH22, GH23 and GH24 lysozymes, as well as GH19 chitinases, all members of the “lysozyme superfamily” [2,6,7,8,9] This list could be extended toward GH80 family based on primary sequence similarities [10]
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