Accessory active site residues of Streptomyces sp. N174 chitosanase

Abstract

The chitosanase from Streptomyces sp. N174 (CsnN174) catalyzes the hydrolysis of b-1,4-glycosidic links in chitosan, a water-soluble derivative of chitin composed of d-glucosamine (GlcN) with a variable but minor proportion of N-acetyl-d-glucosamine (GlcNAc) [1]. Research on the enzymatic hydrolysis of chitosan is driven by the fact that this polymer has numerous potential applications and that its properties often depend on its molecular mass [2]. CsnN174 belongs to family 46 of the glycoside hydrolases (GH46), endohydrolase-type enzymes acting via an inverting mechanism [3,4]. GH46 enzymes belong to the GH-I clan [5] together with lysozymes from family GH24 (the most studied being the lysozyme from T4 phage). Enzymes from these two families share the same catalytic mechanism and are folded similarly, with two globular The chitosanase from Streptomyces sp. N174 (CsnN174) is an inverting glycoside hydrolase belonging to family 46. Previous studies identified Asp40 as the general base residue. Mutation of Asp40 into glycine revealed an unexpectedly high residual activity. D40G mutation did not affect the stereochemical mechanism of catalysis or the mode of interaction with substrate. To explain the D40G residual activity, putative accessory catalytic residues were examined. Mutation of Glu36 was highly deleterious in a D40G background. Possibly, the D40G mutation reconfigured the catalytic center in a way that allowed Glu36 to be positioned favorably to perform catalysis. Thr45 was also found to be essential. Thr45 is thought to orientate the nucleophilic water molecule in a position to attack the glycosidic link. The finding that expression of heterologous CsnN174 in Escherichia coli protects cells against the antimicrobial effect of chitosan, allowed the selection of active chitosanase variants after saturation mutagenesis. Thr45 could be replaced only by serine, indicating the importance of the hydroxyl group. The newly identified accessory catalytic residues, Glu36 and Thr45 are located on a three-strand b sheet highly conserved in GH19, 22, 23, 24 and 46, all members of the 'lysozyme superfamily'. Structural comparisons reveal that each family has its catalytic residues located among a small number of critical positions in this b sheet. The position of Glu36 in CsnN174 is equivalent to general base residue in GH19 chitinases, whereas Thr45 is located similarly to the catalytic residue Asp52 of GH22 lysozyme. These examples reinforce the evolutionary link among these five GH families.Abbreviations (GlcN) n , b-D-glucosamine oligosaccharide with n monomer units; CsnN174, chitosanase from Streptomyces sp. N174; GH, glycoside hydrolase family; GlcN, D-glucosamine; GlcNAc, N-acetylglucosamine.

Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01

“…As described previously [10], the role of Asp 43 in the inverting hydrolysis reaction is to function as a base and activate a key water molecule that can attack the C-1 carbon in the -1 subsite. In the structure of D43A OU01 the nucleophilic water molecule is still present as shown in Figure 1D, and retains its interaction with Thr 48 (Thr 45 in N174), which confirms that this residue is important to orient the water for nucleophilic attack [10,41]. However, because the side chain of Asp 43 is missing, this water cannot be activated to attack the glycosidic bond efficiently.…”

“…chitosanase N174 (Asp 40 of N174 is equivalent to Asp 43 in chitosanase OU01) [41]. This was hypothesized to result from a reconfiguration of the catalytic center that repositioned Glu 36 (Glu 39 in OU01) to perform catalysis [41].…”

“…This was hypothesized to result from a reconfiguration of the catalytic center that repositioned Glu 36 (Glu 39 in OU01) to perform catalysis [41].…”

A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding

“…In order to facilitate the saturation mutagenesis procedure, derivatives of the previously described pUC19-csnN174 and pUC19-csnN174-D40G plasmids have been generated [17]. These derivatives, named pUC19-csnN174-AN and pUC19-csnN174-D40G-AN, harboured two unique restriction sites Nco I and Age I , respectively upstream and downstream from the Arg42 codon.…”

“…Wild-type, D40G, R42E, R42K, D40G+R42E and D40G+R42K chitosanases were purified from recombinant S. lividans TK24 culture supernatants as previously described [17]. Chitosanase and protein assays were as described [21].…”