Oligosaccharide hydrolysis by chitosanase enzymes monitored by real-time electrospray ionization–mass spectrometry

Dennhart, Nicole; Fukamizo, Tamo; Brzeziński, Ryszard; Lacombe-Harvey, Marie-Ève; Letzel, Thomas

doi:10.1016/j.jbiotec.2008.02.004

Cited by 35 publications

(24 citation statements)

References 24 publications

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“…Although (to our best knowledge) a GH19 enzyme with a retaining reaction mechanism has not been disclosed in the literature, it was worth trying to introduce analogous mutations in CsnN174, considering the similarity of the structural cores among GH46 and GH19 enzymes [8]. We thus mutated Asp40 of chitosanase into Gly [15]. In preliminary studies, the D40G mutant revealed significant activity, unexpected for an enzyme devoid of its general base catalytic residue.…”

Section: N174 Chitosanase Devoid Of Asp40 Retains Significant Enzymatmentioning

confidence: 99%

“…The reaction time course of D40G mutant enzyme was thus investigated with (GlcN) 5 and (GlcN) 4 substrates and monitored by real-time MS [15]. As shown in Fig.…”

Section: N174 Chitosanase Devoid Of Asp40 Retains Significant Enzymatmentioning

confidence: 99%

“…5A). Glu36 appears to be a minor player in the wild-type configuration, because its substitution by Asp, Asn, Gln or even Ala had minor effects on activity, decreasing the catalytic constant at most by one third and slightly increasing the K m value (A) and (C) were adapted from Dennhart et al [15] with permission. Fig.…”

Section: Glu36 As a Possible Alternative General Base Residuementioning

confidence: 99%

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Accessory active site residues of Streptomyces sp. N174 chitosanase

et al. 2009

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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.

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Section: N174 Chitosanase Devoid Of Asp40 Retains Significant Enzymatmentioning

confidence: 99%

“…The reaction time course of D40G mutant enzyme was thus investigated with (GlcN) 5 and (GlcN) 4 substrates and monitored by real-time MS [15]. As shown in Fig.…”

Section: N174 Chitosanase Devoid Of Asp40 Retains Significant Enzymatmentioning

confidence: 99%

Section: Glu36 As a Possible Alternative General Base Residuementioning

confidence: 99%

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Accessory active site residues of Streptomyces sp. N174 chitosanase

et al. 2009

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“…Mass spectrometry makes such labeling redundant and allows sensitive detection of various molecules as protonated or deprotonated ions or charged salt-adduct ions (Kalo et al, 2006). In previous studies, we reported the mass spectrometric observation of enzymatic activity and reaction time-courses by continuous flow ESI-MS using hen egg white lysozyme (HEWL) (Dennhart and Letzel, 2006) and two types of chitosanases (endo and exo) (Dennhart et al, 2008). In these studies, the ions of the substrate, the intermediate products, as well as the final products from the oligosaccharide substrates were simultaneously detectable without labeling.…”

Section: Introductionmentioning

confidence: 99%

26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

Dennhart

Weigang

Fujiwara

et al. 2009

Journal of Biotechnology

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“…With an increasing interest in chitooligosaccharides for their neutraceutical, physiological and therapeutic benefits, an alternative method is needed. An obvious approach to chitosan oligosaccharide production is an enzymatic route, e.g., chitinases to hydrolyse the chitin polymer's ␤-linkages (Aam et al, 2010;Bhattacharya et al, 2007;Haebel et al, 2007;Li and Li, 2009); N-deacetylases to deacetylate GlcNAc groups and produce chitosan Tsigos et al, 2000) and chitosanases (Dennhart et al, 2008;Kuroiwa et al, 2003Kuroiwa et al, , 2008 to produce oligomers with increased solubility. Biocatalyzed chitosan production provides an alternative to thermochemical treatment that is environmentally friendly and that can, potentially, result in selective production of chitosan oligomers of defined acetylation/deacetylation pattern.…”

mentioning

confidence: 99%