Role of acidic amino acid residues in chitooligosaccharide-binding to Streptomyces sp. N174 chitosanase

Katsumi, Tomomi; Lacombe-Harvey, Marie-Ève; Tremblay, Hugo; Brzeziński, Ryszard; Fukamizo, Tamo

doi:10.1016/j.bbrc.2005.10.157

Cited by 27 publications

(21 citation statements)

References 19 publications

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“…Since the residues W28 and A30 are localized to the hinge region of chitosanase, we may conclude that binding of the product is likely to proceed through a similar induced-fit steps as binding of the substrate. The "hooked" peak shift of W28 indicates the second product molecule binding to the closing enzyme, which is in accord with our ITC results (Supplementary Table 1) and the literature reports (Katsumi et al 2005). To allow for a more accurate determination of quantitative parameters of the substrate binding mechanism in CsnN174, we performed fitting of the experimental NMR HSQC spectra of the titration of protonated E22A chitosanase with (GlcN)6 using the two-dimensional NMR line shape fitting software TITAN (Waudby et al 2016).…”

Section: Resultssupporting

confidence: 92%

“…Since most polysaccharide hydrolases have a longextended binding cleft, oligosaccharide ligands frequently used for enzymatic analysis of the enzymes may bind to the binding cleft in multiple ways resulting in complicated binding isotherms. For example, two different binding sites were identified in CsnN174 for the chitosan trimer, (GlcN)3, by fluorescence quenching experiments (Katsumi et al 2005).…”

Section: Introductionmentioning

confidence: 99%

“…A similar conformational change was also reported in a family GH19 chitinase from rye seeds (Ohnuma et al 2013), indicating a close relationship between the mechanisms of GH46 and GH19 enzymes . Ligand interactions coupled to conformational changes in protein structures may be sensitively resolved using nuclear magnetic resonance (NMR) line shape analysis (Agafonov et al 2014;Günther and Schaffhausen 2002;Kaplan and Fraenkel 1980;Kern et al 1995;Mittag et al 2006Mittag et al , 2003Rao 1989). In this report we analyzed NMR line shapes in titrations of CsnN174 chitosanase with substrate and product molecules to illuminate the mechanism of coupling between ligand binding and the conformational change in the protein structure.…”

Section: Introductionmentioning

confidence: 99%

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NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

et al. 2017

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Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.

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Section: Resultssupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

et al. 2017

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“…It should be expected that not all binding-site residues contribute equally to a protein's function. Experimentally, it has been observed for several binding sites (23)(24)(25)(26) that the different residues comprising each site can contribute vastly different amounts to the functional processes of the protein (i.e., catalysis or allosteric response).…”

Section: Resultsmentioning

confidence: 99%

Functional residues serve a dominant role in mediating the cooperativity of the protein ensemble

Liu

Whitten

Hilser

2007

Proc. Natl. Acad. Sci. U.S.A.

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Conformational fluctuations in proteins have emerged as a potentially important aspect of biological function, although the precise relationship and the implications have yet to be fully explored. Numerous studies have reported that the binding of ligand can influence fluctuations. However, the role of the binding site in mediating these fluctuations is not known. Of particular interest is whether in addition to serving as structural scaffolds for recognition and catalysis, active-site residues may also play a role in modulating the cooperative network. To address this question, we employ an experimentally validated ensemble-based description of proteins to elucidate the extent to which perturbations at different sites can influence the cooperative network in the protein. Applying this method to a database of test proteins, it is found statistically that binding sites are located in regions most able to affect the cooperative network, even for cooperative interactions between residues distant to the binding sites. This indicates that the conformational manifold under native conditions is determined by the network of cooperative interactions within the protein and suggests that proteins have evolved to use these conformational fluctuations in carrying out their functions. Furthermore, because the energetic coupling pattern calculated for each protein is robust and relatively insensitive to sequence, these studies further suggest that binding sites evolved in regions of the protein that are inherently poised to take advantage of the fluctuations in the native structure.COREX analysis ͉ energetic coupling ͉ native state ensemble ͉ thermodynamic linkage B iological work in living organisms is mediated primarily through the use of protein molecules. Knowledge of the means by which proteins facilitate this work is of paramount importance to an understanding of function as well as the diseases that result from aberrant function. Of particular interest in recent years has been the observation that proteins are highly dynamic molecules that experience dramatic conformational excursions from the canonical high-resolution structure (1-3). In addition, it has also become clear that conformational dynamics play an important role in determining the ability of proteins to perform such diverse tasks as catalysis, allosterism, and signal transduction (4-6).The observation that protein conformational fluctuations play an important role in function suggests that functional sites in proteins may be uniquely coupled to structural fluctuations (7-9) and thus identifiable by how perturbations at these sites affect the conformational manifold. Here we use an ensemblebased model of the protein to explore this issue. By identifying a statistically significant difference in the response of the ensemble to perturbations at active sites of proteins, as determined from a representative test set, we find that binding sites play a unique role in influencing the cooperative network within proteins. This suggests that binding sites have been pre...

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“…Chitosanase and protein assays were performed as described previously [37]. All chitosanase forms were purified from recombinant S. lividans TK24 culture supernatants as described previously [10] except that the gel-filtration step was replaced by the more rapid hydroxyapatite chromatography [15].…”

Section: Chitosanase Purification and Assaymentioning

confidence: 99%

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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Role of acidic amino acid residues in chitooligosaccharide-binding to Streptomyces sp. N174 chitosanase

Cited by 27 publications

References 19 publications

NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

Functional residues serve a dominant role in mediating the cooperativity of the protein ensemble

Accessory active site residues of Streptomyces sp. N174 chitosanase

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